bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1066: Monoamine Oxidase A And Organic Cation Transporter 3 Coordinate Intracellular Adrenergic Signaling To Calibrate Cardiac Function
Authors: Wang Ying , Zhao Meimi , Xu Bing , Xiang Yang K
Published: November 2022

Aims:
We have recently identified a functional pool of intracellular β
1
adrenergic receptors (β
1
ARs) crucial for cardiac function. However, the systematic signaling network of intracellular β
1
ARs remains elusive. Here, we aim to uncover the integrative control of cardiac function, compartmentalized β
1
AR signaling, and potential interface between two critical modulators of catecholamine homeostasis, MAO-A and OCT3.

Methods and Results:
Cardiac deletion of MAO-A (MAO-A-CKO) elevated myocardium catecholamines, cAMP levels and cardiac ejection fraction by disrupting intracellular catecholamine degradation. Deletion and pharmacological inhibition of MAO-A both enhanced cardiac adrenergic response, with elevated ejection fraction and heart rate. Inhibiting OCT3 by corticosterone prevented the impacts of MAO-A inhibition on cardiac function. Using subcellular anchored Förster resonance energy transfer (FRET) biosensors, we then determined the regulation of compartmentalized β
1
AR/PKA signaling at the sarcoplasmic reticulum (SR) and plasma membrane (PM) microdomains. We also examined local PKA-substrates phosphorylation and myocyte excitation-contraction coupling. MAO-A deletion and inhibitor (MAOi) enhanced local PKA activity at the SR, phosphorylation of phospholamban, Ca
2+
cycling, and contractile response. Whereas overexpression of MAO-A impairs SR-localized PKA activity and phospholamban phosphorylation. In contrast, these modifying of MAO-A did not affect PKA activity at PM and L-type Ca
2+
channel phosphorylation. Inhibiting OCT3 blocked blocking catecholamine transport and diminished the efficacy of MAO-A inhibitor (MAO-A) and MAO-A-CKO to regulate SR-localized β
1
AR signaling. In parallel, OCT3 inhibition and deletion prevented the MAO-A-dependent enhancement of Ca
2+
cycling and cardiac contractile function.

Conclusion:
Together, we show that MAO-A and OCT3 act in a concert to fine-tune the intracellular catecholamines, activation of SR-localized β
1
AR-PKA signaling, and cardiac fight-or-flight response. Further, we reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of those drugs in cardiac implications.
Abstract P1067: IRS Mediated Recruitment Of PI3Kgamma By Insulin Inhibits Beta-adrenergic Receptor Function
Authors: Sahu Anita , Mukherjee Sromona D , Witherow Conner , Stenson Kate , Tesmer John , Mohan Maradumane L , Naga Prasad Sathyamangla V
Published: November 2022

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.
Abstract P1069: Integrin Linked Kinase Controls Human Valve Endotelial Cells Pro-osteogenic Activation In A TFG-b/NO Dependent Manner
Authors: Sanchez Sandra , Castro Mercedes , Cook Calvete Alberto , Delgado Marín María , Hernandez Navarro Ignacio , Lopez-Menendez Jose , Zaragoza Carlos , Saura Marta
Published: November 2022

Calcific aortic valve disease (CAVD) is expected in the aging population. CAVD physiopathology arises from endothelial dysfunction leading to lipid accumulation, inflammation, and osteogenic transformation. Many studies suggest that valve endothelial cells (VECs) undergoing EndMT may differentiate into osteoblastic interstitial cells. We previously showed that Integrin linked kinase (ILK) regulates vasomotor function by preventing endothelial nitric oxide synthase uncoupling. ILK expression in vascular endothelium plays an essential cardioprotective role. Moreover, we have found a significant negative correlation between human valve protein levels of ILK and the osteogenic marker Runx2 and BMP2 in 70 patients with calcific aortic stenosis, a higher decrease at the endothelial level. We hypothesized that Endothelial ILK plays a protective role in human valve endothelial cells osteogenic transition. Isolation of human valve endothelial cells from the aortic (aortic hVECs) and ventricular sides (ventricular hVECs) show a specific ILK decrease in the aortic hVECs from calcific compared to non-calcified valve (p<0.05). Silencing ILK expression in hVECs decreased NO production and increased ROS (Reactive Oxygen Species) production as early as 72 hours. After five days of silencing ILK, hVEC express myofibroblast markers SM22a, α-SMA, MMP12, MMP9, and SNAI 1 while losing the endothelial markers (CD31, and vWF (p<0.01). ILK silenced hVECs (siILK-hVECs) express the osteogenic marker RunX2 indicating osteogenic differentiation and developed calcified nodules. Cell treatment with Noggin, a BMP2 antagonist, did not reverse the increase of Runx2, thus excluding the implication of the BMP2/RUNX2 axis. However, silk-hVECs showed an increase in phosphorylated-SMAD 2, suggesting a TFG-β -dependent mechanism. Treatment of siILK-hVECs with the NO donor DETA-NO decreased Smad 2 activation and RunX2. Moreover, DETA-NO treated siILK hVECs cultured in pro-osteogenic media did not develop calcification. Collectively, our results point to a crucial role of ILK in keeping human valve endothelial cell phenotype preventing valve calcification in a Nitric Oxide-TFGβ1 dependent manner. Financed by the Regional European fund “a way to achieve Europe.” JCCM SBPLY/19/180501/000055S and ISCIII PI20/00930
Abstract P1071: Concurrent Stat1 And Stat3 Signaling Modulates Tlr4/nfkb-mediated Immunomodulatory And Antifibrotic Effects Of Cardiomyocyte-damaged Released S100a1 Protein In Cardiac Fibroblasts
Authors: Busch Martin , Daszenies Michael , Most Patrick
Published: November 2022

Background:
We previously showed that S100A1 released from damaged cardiomyocytes can accelerate post-myocardial infarction in vivo healing. The action of S100A1 as a damage-associated molecular pattern seemed to occur via an immunomodulatory (e.g., IL10 increase) and antifibrotic impact (e.g., COL1A1 and ACTA2 downregulation) in cardiac fibroblasts (CFs) via a TRL4/NKFB pathway. The signaling network in CFs conveying this effect has not been characterized yet.

Methods and Results:
We performed a 48h time-resolved transcriptome analysis on cultured adult rat CF stimulated with rec. human S100A1 protein (10
-6
M). Bioinformatic GSEA yielded an instant and almost exclusive significant upregulation of the chemokine activity GO-term with a delayed suppression of ECM genes. A transcription factor enrichment analysis for the differentially regulated transcripts indicated an immediate activation of NFKB/RELA with a concurrent temporary STAT1 but lasting STAT3 signaling. A secretome LC-MS/MS-based analysis confirmed the shift of S100A1-treated CFs to mostly chemokine secreting cells. With CCL2 as most secreted chemokine and COL1A1 and ACTA2 as established antifibrotic marker genes for released S100A1, we used a RT-PCR readout for these genes in response to siRNA-based knock-down (KD) of STAT1 and STAT3 in S100A1-treated CFs, while TLR4 KD was used to abrogate NFKB/RELA signaling due to known mechanisms (n=10 each group). S100A1 treatment upregulated CCL2 (16-fold*) and downregulated COL1A1 (3.1-fold*) and ACTA2 (2.9-fold*) mRNA expression vs control (*P<0.01) that was blunted by TLR4 KD. STAT3 KD doubled the CCL2 mRNA increase (32.9-fold*), attenuated COL1A1 mRNA downregulation by S100A1 treatment to 2-fold* but left ACTA2 downregulation unaffected vs contr. (*P<0.05). Conversely, STAT1 KD completely blocked the S100A1 mediated downregulation of ACTA2 but neither impacted S100A1-mediated changes in CCL2 nor COL1A1.

Conclusion:
Our study identifies concurrent STAT1 and STAT3 activation as a modulator of S100A1-mediated TLR4-NFKB signaling in CFs. As S100A1-activated CFs express e.g., IL-10, we speculate that IL-gp130 trans signaling may convey this effect guiding further studies on S100A1’s extracellular effects in the diseased heart.
Abstract P1077: Nicotine Compromises Metabolic Adaptation Of The Heart To Injury By Altering PHLPP Isoforms
Authors: Mohammed Abdul Khaja Shameem , Wilson Cekia , Purcell Nicole
Published: November 2022

Rationale:
Cardiovascular (CV) disease remains the leading cause of death and disability in the US and worldwide and tobacco use is a major risk factor for increasing the incidence of disease. Consequently, there is a need for understanding the cardiovascular impact of systemic nicotine on cardiac physiology and heart failure progression following injury. We have demonstrated that removal of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) increases Akt activity and protects the heart from oxidative damage, however downregulation of PHLPP2 causes damage. Preliminary data suggest that exposure to nicotine increases PHLPP1 and decreases PHLPP2 levels in the heart. Therefore, linking the direct pathogenic effects of nicotine on these cell-survival intracellular pathways such as those that utilize PHLPP1 activity needs to be rigorously examined for therapeutic efficacy and intervention.

Hypothesis:
We hypothesize that exposure to high levels of nicotine will compromise the heart and increase susceptibility to ischemic injury by altering PHLPP isoform expression.

Methods:
Using
in vivo
systems we investigated the effect of nicotine exposure on PHLPP expression and cardiomyocyte function and injury. Male and female wild-type WT (n=4) and PHLPP1 KO (n=4) mice were implanted with osmotic minipumps containing nicotine (5mg/kg/day) or saline for 7days and changes in protein and gene expression were examined. A second cohort of WT animals was exposed to nicotine (7 days) and subjected to 1 hour ischemia and 24 hours reperfusion (n=6).

Results:
Following 7 days of nicotine exposure, we found PHLPP1 protein expression was significantly increased (1.7 ± 0.25 fold) and PHLPP2 significantly decreased (0.8 ± 0.04 fold) in the hearts of WT mice. However, in PHLPP1 KO mice, nicotine exposure significant increased PHLPP2 levels in the heart (1.5 ± 0.044fold). Also, we found that exposure to nicotine suppressed genes important for mitochondrial biogenesis and decreased antioxidant protein expression in the hearts of WT mice while antioxidant signaling was significantly increased in KO hearts. Following 7 days of nicotine exposure WT mice have increased infarct injury (39% ± 3.2, n=6) following 24-hour reperfusion compared to saline treated WT (31% ± 0.55, n=4) with the same area at risk. Preliminary studies found that PHLPP1 KO significantly protected the heart from ischemic injury (26% ± 0.38, n=3), however the effect in the presence of nicotine is under investigation.

Conclusions:
Our data suggests that nicotine exposure has detrimental effects on the heart by altering PHLPP isoform expression which compromises oxidative signaling and increases injury. Further studies will investigate the mechanism of nicotine induced PHLPP isoform expression both
in vitro
and
in vivo
and determine whether targeting PHLPP1 could significantly impact cardiac outcome associated with nicotine use.
Abstract P1079: S100A1-SUMO Interaction Via SUMO Interaction Motif (SIM) Of The S100A1 C-terminus Domain Is Critical For S100A1 Post-translational Protein Stability
Authors: Jebessa Zegeye Hailu , Glaser Manuel , Schneider Andrea , Zhao Jemmy , Seenivasan Ramkumar , Busch Martin , Wade Rebecca , Most Patrick
Published: November 2022

Background & Objectives:
S100A1 protein is a 10 kDa Ca
2+
sensor & abundantly expressed in cardiomyocytes (CMs) of vertebrates. Marked reduction in S100A1 level is hallmark of diseased heart, vice versa, addition of S100A1 by gene transfer rescues diseased heart contractile dysfunction. Since our understanding of the molecular circuits that may contribute to the regulation of S100A1 protein levels are still scarce, we conducted a hybrid computational structural & experimental approach to unveil underlying molecular residues & mechanisms that may control S100A1’s protein stability.

Methods & Results:
We employed in silico computational prediction & molecular docking tools, respectively, to inform experimental approaches in order to characterize residues within the 94 amino acid (aa) of S100A1. A web server-based GPS-SUMO 2.0 analysis of the human S100A1 sequence unveiled a putative SUMO interacting (SIM) motif (76-VVLVA-80) within the alpha-helical C-terminus of S100A1. Restrained docking with HADDOCK predicted a molecular interaction between SUMO-1 & the SIM-lined groove of the Ca2+-bound (holo) S100A1 homodimer that presents a potential novel type of interaction mode. We then performed an in vitro S100A1-SUMO interaction assay in the presence of 1 mM Ca
2+
or 1 mM EGTA & the assay revealed calcium dependent specific S100A1-SUMO proteins interaction. Overexpression of S100A1 together with SUMO1 increases S100A1 protein abundance in CMs & COS1 cells without changing the mRNA level of S100A1. Overexpression assays in COS1 cells & CMs involving S100A1 truncation mutant lacking SIM motif (S100A1-1-74) or site directed mutagenesis deleted (DSIM) or Alanine replaced SIM of S100A1 showed that S100A1 lacking SIM motif either via truncation & deletion or Alanine substitution led to, respectively, absence of detection or massively reduced overexpression of S100A1 protein without affecting the mRNA overexpression of the mutants. The aforementioned mutants could be rescued at protein level in CMs & COS1 cells by addition of proteasome inhibitor-MG-132.

Conclusion:
Here we describe a yet unrecognized post-translational molecular checkpoint for S100A1’s protein stability involving a SIM-mediated interaction between S100A1 & SUMO
Abstract P1084: Mature Ipsc-derived Atrial Cardiomyocytes Show That Saturated Fatty Acids Differentially Modulate Atrial Remodeling In Obesity-induced Atrial Fibrillation Compared To Mono And Poly-unsaturated Fatty Acids.
Authors: Sridhar Arvind , Ly Olivia T , Arif Pavel Mahmud , Chen Hanna , Gonzalez Francisco , Perike Srikanth , Hong Liang , McCauley Mark , Rehman Jalees , Darbar Dawood
Published: November 2022

Background:
Increased free fatty acids (FFA) and FA infiltration, both major hallmarks of obesity can individually modulate changes leading to AF. For example, mass spectrometry on obese mouse heart tissues have shown that saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) are all significantly increased compared to controls. The highest increase was shown to be in linoleic acid (LA), a PUFA followed by palmitic acid (PA, SFA) and oleic acid (OA, MUFA). Although reports suggest that some FAs induce ventricular arrhythmias in mice, the differential electrophysiological effects of SFAs, MUFAs, and PUFAs on the atria remain unclear.

Objective:
To determine the individual roles of SFAs, MUFAs, and PUFAs in mediating atrial remodeling in obesity-induced AF using mature human induced pluripotent stem-derived atrial cardiomyocytes (iPSC-aCMs).

Methods:
We compared iPSC-aCMs and HL-1 cells treated with bovine serum albumin (BSA) with palmitic acid (PA), oleic acid (OA) and LA cells (600 uM each) for 24 hours and 1 week. Ion channel function were assessed using optical voltage mapping and whole-cell patch clamping, and we assessed atrial remodeling using qPCR, and western blotting.

Results:
iPSC-aCMs treated with PA, OA, and LA displayed higher expression of
FABP3
and
CPT1A
suggesting increased FA metabolism (Figure A-C). In HL1 cells, protein expression of Cpt1a was increased in all groups after FA treatment (Figure D). PA differentially modulated expression of Kv7.1 and NCX1 in HL1 cells (Figure E-F) and ion channel mRNA expression in iPSC-aCMs (Figure G-L). After 1 week PA and LA treatment b-adrenergic signaling was significantly increased in iPSC-aCMs while OA decreased it compared to controls (Figure M-O). Lastly, 24-hour PA, OA, LA treatment differentially modulated the atrial action potential (AP). While PA and LA significantly shorten the action potential, there was no significant changes to the AP after OA treatment.

Conclusions:
Using mature iPSC-aCMs, we showed for the first time that SFA, MUFA, and PUFA not only increased FA metabolism but differentially affected b-adrenergic signaling and ion channel remodeling leading to differential effects on the atrial AP. Our findings may have important implications for the management of obesity-induced AF in patients.
Abstract P1086: Lrrc10 Is A Multifaceted Modulator Of Cardiac Calcium And Sodium Channels
Authors: Del Rivero Morfin Pedro J , Chakouri Nourdine , Ben Johny Manu
Published: November 2022

L-type calcium channels (LTCC) are vital for heart physiology as they allow entry of calcium to the cardiomyocyte, ultimately leading to contraction. These channels are exquisitely modulated by auxiliary subunits and a vast array of regulatory proteins. Heart-specific leucine-rich repeat-containing protein 10 (Lrrc10) plays a key role as a regulator of LTCC. Previous work has shown that Lrrc10 knockdown in zebrafish disrupts development of the cardiac tube, while ablation of Lrrc10 in mice results in heart dysfunction and maladaptation to increased cardiac afterload. Moreover, human mutations in Lrrc10 gene have been linked to dilated cardiomyopathy, and sudden unexplained nocturnal death syndrome (SUNDS). Still, the molecular mechanisms underlying Lrrc10 regulation of LTCC is not fully established. Beyond this, it is unknown whether Lrrc10 modulates other cardiac ion channels. Here, we hypothesized that Lrrc10 serves as a versatile regulator of four-domain voltage-gated ion channels, while mutations in Lrrc10 differentially disrupt channel regulation. Using whole-cell and single-channel electrophysiology, we found that Lrrc10: 1) upregulates LTCC currents by increasing peak open probability; 2) accelerates calcium-dependent inactivation; and 3) evokes a hyperpolarizing-shift in voltage-dependence of activation. Flow cytometric FRET 2-hybrid assay showed that Lrrc10 interacts with LTCC cytoplasmic subunits, specifically the N- and C-termini. We further used FRET 2-hybrid analysis to evaluate the interaction of Lrrc10 with related cardiac ion channels. We observed robust Lrrc10 interaction with Na
V
1.5 channels. Multi-channel electrophysiology recordings revealed increased late sodium currents, suggesting that Lrrc10 also modulates Na
V
1.5 activity. Additionally, electrophysiological studies showed that Lrrc10 variants linked to dilated cardiomyopathy and SUNDS disparately alter modulation of calcium and sodium channels. In conclusion, Lrrc10 is a multifaceted regulator of four-domain, voltage-gated cardiac ion channels, a finding that will lend critical insights into heart function and disease.
Abstract P1087: P21-activated Kinase 1 Regulates Intrinsic Pacemaker Activity Through HCN4 Expression
Authors: Pereira Carlos H , Banach Kathrin
Published: November 2022

Introduction:
Cardiac pacemaker activity generated in the sinoatrial node (SAN) depends on a coupled clock mechanism that consists of a calcium (Ca-) and a voltage clock (V
m
-clock). Attenuation of either clock results in bradycardia which increases the risk for atrial fibrillation (AF). We have previously demonstrated that p21-activated kinase 1 (Pak1) is a negative regulator of NADPH oxidase 2 dependent production of reactive oxygen species (ROS). Here we hypothesized that attenuated Pak1 activity increases the susceptibility for AF by attenuating SAN function.

Methods:
In wild type (WT) and Pak1 knock-out (Pak1
-/-
) mice (FVBN), ECGs and atrial electrograms were recorded in vivo and in isolated Langendorff (LD)-perfused hearts.

Results:
Pak1
-/-
compared to WT mice exhibited an attenuated intrinsic HR in vivo after autonomic blockage with atropine (1 mg/kg) and propranolol (1 mg/kg) (WT: 399 ± 8.05 bpm, n=16; Pak1
-/-
: 365 ± 9.6 bpm, n=13, p<0.05) and in the isolated heart (WT: 343 ± 8.5 bpm, n=24; Pak1
-/-
: 280 ± 6.03 bpm, n=28, p<0.01), suggesting attenuated SAN impulse generation. Block of the Ca-clock (cyclopiazonic acid (CPA): 5 μM) did not eliminate the difference in HR. Block of the V
m
-clock with the hyperpolarization activated cyclic nucleotide gated channel (HCN4) blocker ivabradine (IVA: 3 μM) however, had an attenuated effect on HR in Pak1
-/-
(WT
IVA
: -45 ± 2.5 %, n=6; Pak1
-/-
IVA
: -22 ± 3.2 %, n=7, p<0.001) and eliminated the difference in SAN frequency (WT
IVA
: 186 ± 17 bpm, n=6; Pak1
-/-
IVA
: 234 ± 15 bpm, n=7) supporting a decreased V
m
-clock function. Attenuated HCN4 expression in Pak1
-/-
could be recovered by suppression of Histone deacetylases class II activity (LMK235: 5 mg/kg/day, 3 days); WT
LMK
: 322 ± 9.4 bpm, n = 5; Pak1
-/-
LMK
314 ± 3.4 bpm, n= 5;) as well as by scavenging of ROS (TEMPOL supplemented drinking water, 2 mmol/L, 3 days; WT
Tem
: 332 ± 12.7 bpm, n = 5; Pak1
-/-
Tem
: 318 ± 22 bpm, n= 5). Both treatments, restored the contribution of V
m
-clock to pacemaker activity in Pak1
-/-
animals (WT
LMK
: -38 ± 8.3%, n = 5; Pak1
-/-
LMK
: -38 ± 1.2%, n= 4; WT
Tem
: -46 ± 3.7 %, n = 5; Pak1
-/-
Tem
: -41 ± 2.5 %, n= 5) and attenuated AF inducibility.

Conclusion:
In both sexes attenuated Pak1 signaling results in SAN bradycardia by ROS/HDAC dependent suppression of HCN4 expression.
Abstract P1088: Polycystin-1 Protects Against Experimental Atrial Fibrillation
Authors: Hendrickson Troy , Perez William , Giese Abigail , Altamirano Francisco
Published: November 2022

Atrial fibrillation (AF) is the most common cardiac arrhythmia with a 1 in 4 lifetime risk. AF impairs cardiac function and increases the risk of stroke and heart attack. Although this is a common and harmful disease, little is known about the molecular mechanisms driving this disease. We have previously demonstrated that the protein polycystin-1 (PC1) plays a role in atrial cardiomyocyte excitation-contraction coupling in ventricular cardiomyocytes. Interestingly, patients with mutations in PC1 encoding gene have increased rates of atrial arrhythmias, including atrial fibrillation. Here, we sought to study the role of PC1 in atrial cardiomyocyte function and atrial fibrillation inducibility. We performed intracardiac electrophysiology with burst pacing in two mouse models with impaired PC1 function: 1) a mouse model harboring R3277C mutation (RC/RC) known to cause autosomal polycystic kidney disease, and 2) cardiomyocyte-specific PC1 KO (CKO). We observed increased rates of atrial fibrillation inducibility in both RC/RC and PC1 CKO mice compared to their respective control littermates (76.9% in RC/RC and 81.8% in CKO vs 20.0% and 11.1% in their respective WT controls). To elucidate molecular mechanisms driving increased susceptibility to atrial fibrillation, we determined whether PC1 affects excitation-contraction coupling in atrial cardiomyocytes. Human induced pluripotent stem cells-derived atrial cardiomyocytes (hiPSC-aCM) were treated with control or PC1 siRNA. Action potentials and intracellular calcium were measured using Fluovolt and Fluo-4, respectively. PC1 ablation decreased action potential duration and calcium transient peak amplitude elicited by electrical stimulation in hiPSC-CM. Decreased calcium transient peaks were also observed in HL-1 atrial cell line treated with siRNA and adult atrial cardiomyocytes isolated from RC/RC mice compared to their control counterparts. Unexpectedly, RNA-sequencing revealed altered DNA Damage Response gene expression. DNA damage has been implicated in the pathogenesis of atrial fibrillation. Therefore, we studied whether tachypacing elicited DNA damage in hiPSC-aCM. We observed increased levels of H2AX phosphorylation (gamma H2AX) in hiPSC-aCM with reduced PC1 expression. Our data show that PC1 mutations increase atrial fibrillation inducibility in multiple mouse models, likely due to impaired DNA damage response and altered action potential and calcium handling.
Abstract P1092: Voltage Gated Sodium Channel Subunit Mimetic Peptide βadp1 Increases Regulated Intramembrane Proteolysis Of Scn1b/β1
Authors: Williams Zachary J , Hoagland Daniel , Jourdan Jane , Gourdie Robert G
Published: November 2022

Clinical treatment for cardiac arrhythmias based on targeting ion channels is limited and can have deadly side effects. We previously reported that acute administration of βadp1 resulted in decreased action potential conduction velocity and increased arrhythmias in isolated guinea pig hearts. These changes were associated with disruption of trans-homophilic adhesion of the β1 subunit within gap junction-adjacent perinexal domains in intercalated discs. Using Electric Cell-Substrate Impedance Sensing (ECIS), we found that βadp1 shows opposing acute and long-term effects on β1 mediated adhesion. βadp1 decreases β1 mediated adhesion in 1610 cells stably expressing SCN1B/β1 (1610β1) over the first 24 hours, but after 48 hours is associated with increased adhesion. Regulated Intramembrane Proteolysis (RIP) of β1 sequentially by BACE-1 and γ-secretase results in a cleaved intracellular fragment that translocates to the nucleus and alters transcription of various genes, including VGSC subunits. Therefore, we hypothesized the long-term gain-of-adhesion-function with βadp1 treatment resulted from increased RIP of β1, with downstream VGSC gene expression effects. Inhibition of γ-secretase with DAPT accumulates a C-terminal fragment (CTF) of β1 during RIP, enabling quantification of changes to RIP. Thus, 1610β1 cells were treated with 50μM βadp1, 1μM DAPT, 50μM βadp1+1μM DAPT or 0.1% DMSO control. Cells were fixed for β1 immunofluorescence (IF) or lysate was collected at 6-, 24-, and 48-hours post treatments for Western blotting. DAPT treatment significantly increased CTF levels compared to control at all timepoints (p<.0001). However, βadp1+DAPT treatment resulted in greater accumulation of β1 CTF levels over DAPT alone at each timepoint. (n=6, 6 hr: p<.01, 24 hr: p<.01, 48 hr: p<.05). βadp1+DAPT treatment caused a significant increase in full-length β1 at 6 hours, but no difference at 24 or 48 hours. The data suggest depletion of β1 by increased RIP of β1 after βadp1 treatment. IF showed an increase in abundance of β1 48 hours after βadp1 treatment compared to DMSO. In sum, our data indicates that βadp1 may upregulate RIP of β1 with potential downstream effects on gene transcription of VGSC subunits, suggesting a path to a novel class of antiarrhythmics.
Abstract P1097: Heart Rate Variability Analysis In Rodents Reveals Altered Cardiac Autonomic Regulation Following Myocardial Infarction
Authors: Pizzo Emanuele , Berrettoni Silvia , Kaul Ridhima , Cervantes Daniel O , Di Stefano Valeria , Singh Kanwardeep , Jain Sudhir , Jacobson Jason , Rota Marcello
Published: November 2022

Heart rate variability (HRV) is an index of cardiovascular health and reflects the ability of the heart to modify beating rate in response to neurohumoral factors. Alterations of HRV have been reported in patients with myocardial infarction (MI), but whether experimental models of ischemic disease recapitulate features of HRV observed in human remains to be clarified. HRV was evaluated in female mice using electrocardiograms collected in the conscious, restrained state using a tunnel device. Naïve mice and animals after permanent coronary artery ligation (MI) were studied. HRV was assessed using time-domain, frequency-domain, and non-linear parameters. By echocardiography, all infarcted animals presented akinetic anterior LV free wall and, respect to naïve mice, a 1.8-fold increase in LV diameter and a 64% reduction in ejection fraction. At 3 to ~30 days after MI, RR interval duration was preserved with respect to non-infarcted mice, whereas standard deviation of RR interval duration (SDRR), was reduced by 61-70% at 3 days to ~30 days after MI. Standard deviation of instantaneous (SD1) and long-term (SD2) RR interval variability, indicative of parasympathetic and sympathetic influence, respectively, were derived from Poincaré plots of RR
i
and RR
i+1
intervals. SD1 and SD2 were reduced after MI (64-73% and 61-70%, respectively). Moreover, by frequency-domain analysis, the contribution of high- (1.5-5 Hz), low- (0.15-1.5 Hz), and very low-frequency (0-0.15 Hz) components of RR interval oscillations were assessed. High-frequency components, which are under the influence of the sympathetic nervous system, were not affected after MI; low-frequency oscillations, attributed to baroreflex-mediated modulation of heart rate, were reduced at 3 and 7 days after MI, whereas very low-frequency components, attributed to the modulatory action of the renin-angiotensin system, thermoregulation, and, partly, parasympathetic activity, were reduced from 3-30 days after MI. Moreover, the ratio between low and high frequency components, indicative of sympathovagal balance, was reduced after MI. In conclusion, myocardial infarction in rodent results in altered heart rate variability, which appears to be secondary, in part, to altered autonomic regulation.
Abstract P1098: Atrial Deletion Of AMPK Promotes Atrial Oxidative Stress And The Formation Of Delayed Afterdepolarizations Within The Inter-atrial Pulmonary Vein Junction
Authors: Cacheux Marine , Ma Yina , Wu Xiaohong , Akar Joseph , Young Lawrence H , Akar Fadi G
Published: November 2022

Atrial specific AMPK deletion leads to spontaneous onset of AF. Electrophysiological mechanisms underlying this heightened propensity remain unknown. We hypothesized that AMPK inactivation forms atrial triggered activity leading to atrial ectopy and AF initiation, in large part, by promoting atrial oxidative stress.

Methods:
We performed detailed ex vivo atrial optical action potential (AP) mapping on the posterior surface of hearts from mice with atrial deletion of AMPK a1/a2 (AMPK-dKO) (via Cre recombinase driven by sarcolipin) vs littermate controls (Ctrl), both during steady state pacing and following challenge with right atrial stimulus trains designed to promote calcium overload. Mitochondrial (mito) ultrastructure and markers of atrial oxidative stress were measured. To assess the functional impact of atrial oxidative stress, AMPK-dKO mice underwent chronic
in vivo
treatment with EUK-134 (EUK, 25mg/Kg SQ, 3X/wk) for 16 wks.

Results:
AMPK-dKO (7/11) but not Ctrl (0/5) hearts exhibited subthreshold delayed afterdepolarizations (DAD) that were consistently localized within a discrete inter-atrial region corresponding with the pulmonary veins (PV) junction. These triggers coincided with atrial oxidative stress indexed by a significant (p<0.05) decrease in GSH/GSSG, PRDX3, SOD2, and increase in nitrotyrosine expression along with atrial mito swelling, decreased count and loss of cristae density. Chronic EUK treatment of AMPK-dKO suppressed the incidence (83% to 40%) and frequency (1.81±0.98/s to 0.083±0.08/s) of PV triggers abrogating the genesis of post-pacing atrial AP ectopy originating from the PV (4/5 vs 0/5). Serial in vivo ECG recordings in a subset of AMPK-dKO mice showed reorganization of the RR vs RR+1 relationship following 16 wks of EUK treatment consistent with decreased incidence of premature atrial complexes. Expression of total and S368 phosphorylated Cx43 were both reduced in the atria of AMPK-dKO mice vs Ctrl and restored by EUK reflecting reversal of pathological molecular remodeling by in vivo ROS scavenging.

Conclusions:
AMPK deletion promotes formation of left atrial ectopy from DAD sources within the PV region. AMPK plays a central role in the prevention of atrial oxidative stress and associated arrhythmias.
Abstract P1113: Ploidy Reversal As A Postnatal Developmental Program In Murine Hearts
Authors: Swift Samantha K , Kolell Mary E , Purdy Alexandra L , Flinn Michael A , O'Meara Caitlin C , Rau Christoph D , Patterson Michaela
Published: November 2022

Polyploidization is a normal cellular adaptation for a variety of cell types in mammals, including cardiomyocytes (CMs); however, the development of polyploidization understudied. Recent work suggests that genetics contribute to diverse displays of ploidy in the adult murine heart. Therefore, we hypothesized that the developmental progression to reach differing end-states may similarly be affected. Here, we assessed CM endowment, cell cycle, and ploidy temporally across two diverse inbred mouse strains, A/J and C57Bl/6J. Consistent with previous work, C57Bl/6J hearts displayed rapid cell cycle activation in the first postnatal week largely coinciding with the first round of endomitosis. Total CM numbers are relatively unchanged after P7, while final ploidy states are generally constant from P14 on. In contrast, A/J mice displayed depressed cell cycle activation in the first week of life. Polyploidy in A/Js reaches its peak at P21, whereby only ~3.5% of CMs remained mononuclear and diploid. Interestingly, from 3-6 weeks of age, we observed a dramatic expansion of total CM numbers and of the 1x2N subpopulation to ~8%, which could not be explained by proliferation of the residual diploid population. Instead, the expanded diploid population appears to come from a polyploid CM. This finding was confirmed by analysis identifying a population of CMs which had definitively completed cytokinesis by 6 weeks which was not present at 3 weeks. We believe this is the first report of ploidy reversal by a mammalian CM, a phenomenon first observed by the hepatocyte field. Ongoing single nuclear RNA seq is examining the transcriptomic differences between A/J and C57Bl/6J CMs at P21 to identify this unique “primed” population competent to undergo ploidy reversal. Preliminary results from this study suggest that AJ CM nuclei, regardless of their ploidy, are more likely to be in the cell cycle in comparison to C57. Understanding the developmental paths to end state CM endowment and ploidy states can help us understand the biology of organ size and reciprocally can be applied to stimulating regeneration.
Abstract P1120: Project Singular: Building A Large-scale Genetics Dataset To Fuel Research Towards Curative Solutions For Single Ventricle Heart Disease
Authors: Pickles Diane M , Davis Kaitlin , Keller Kirstie
Published: November 2022

Objective:
Single ventricle (SV) heart disease is a set of rare conditions occurring in 5 out of 100,000 births. Forty years ago, such diagnoses were universally fatal, but surgical advances have enabled most patients to live into their thirties. However, SV patients face a lifetime of expensive, invasive clinical care for complications and co-morbidities and decreased life expectancy and quality of life.

The cause of SV heart disease is still unknown; believed to be both Mendelian and non-Mendelian, multigenic and multifactorial. To date, four genes have been linked to SV disease; however, the penetrance is low, and none are causal. Project Singular aims to uncover the cause(s) of SV by sequencing at least 5,000 SV patients and immediate family members, in conjunction with collecting important medical data, creating the largest genetic dataset available to date in this disease community and make it available free of charge to all qualified researchers.

Methods:
Subjects will be recruited and consented virtually through an online portal and will receive a biospecimen collection kit (saliva or buccal) by mail to return to the Broad Institute of MIT and Harvard. Samples will be processed, quality-controlled, and whole genome-sequenced using Illumina HiSeq at 30X coverage, with methylation arrays conducted on a subset. Sequences will be aligned to the human genome, annotated, deidentified, and securely deposited into the Terra.bio platform. Participants will fill out a survey on demographics and medical history, and patients will provide medical records. Deidentified phenotypic and genotypic data will be made available for free to qualified researchers globally. Integration with other SV-related registries will be undertaken where possible to increase the power of the study.

Conclusion:
Project Singular is the first genetics sequencing initiative of its size and depth focused specifically on SV, the most complex of all congenital heart defects. By enabling patients and families to participate from their homes, Project Singular will reduce barriers of travel and cost to increase diversity of participants. Data democratization of the dataset will fuel discovery in single ventricle etiology and lead to new targeted treatments and potentially curative solutions.
Abstract P1127: Electrical Stimulation For The Maturation Of Induced Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Cook Suh Hee , Twiddy Jack , Daniele Michael , Gluck Jessica
Published: November 2022

Heart disease is the leading cause of death in the U.S. and worldwide. Cardiac tissue engineering offers promising diagnostic tools and treatments. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are an attractive cell source for these tissue engineering applications due to their patient-specificity and theoretical capacity to supply limitless cell quantities. However, these hiPSC-CMs are immature compared to healthy, adult cardiomyocytes, demonstrating immature morphology, metabolics, and electrophysiology. Previous research has shown electrical stimulation shows promise in maturation of hiPSC-CMs.

We aim to create an
in vitro
platform for the electrophysiological maturation of hiPSC-CMs using applied electrical stimulation during the differentiation process. We designed and fabricated a bioreactor for the application of electrical stimulation to hiPSC-CMs in culture. The bioreactor was created by laser cutting two parallel slits into the lid of a 12-well tissue culture plate, through which conductive Toray paper was placed. The space remaining between the slits and the Toray paper was sealed with polydimethylsiloxane (PDMS). Alligator clips were used to connect a function generator to the Toray paper. When sterilized with 70% ethanol, this modified 12-well plate lid can be used to apply electrical stimulation to cells cultured in a 12-well plate, as shown in Figure 1. Future work includes the application of electrical stimulation on day 3 of a well-established Wnti-directed monolayer protocol for the differentiation of hiPSCs into hiPSC-CMs. We expect to observe a resultant improvement in iPSC-CM’s genetic and electrophysiological maturation.
Abstract P1132: Cardiac Tissue Chip Model Of Arteriovenous-fistula-associated Hemodynamics Elicits Fibrotic Changes Seen In Mouse Model
Authors: Graham Caleb , Isayeva-Waldrop Tatyana , Gard William , Ingle Kevin , Lee Timmy , Sethu Palaniappan
Published: November 2022

Introduction:
Cardiovascular disease is the primary cause of death amongst end-stage renal disease (ESRD) patients. Though arteriovenous fistulas (AVFs) are the access of choice for hemodialysis, joining a high-pressure artery and low-pressure vein leads to cardiac volume overload acutely. Debate exists as to the cardiac ramifications of AVFs, due in part to the difficulty of recruiting ESRD patients and controls for prospective studies. Animal models have been essential in AVF studies, but their usefulness is limited by interspecies differences in physiology and other factors. Thus, we have developed a three-dimensional (3D) cardiac tissue chip with tunable pressure and stretch to model the acute hemodynamic changes associated with AVF creation.

Hypothesis:
In this study, we aimed to replicate the hemodynamics of murine AVF models in vitro and hypothesized that if 3D cardiac constructs were subjected to “volume overload”, they would display fibrosis and key gene expression changes seen in AVF mice.

Methods:
Mice underwent either AVF or sham procedure and were monitored for 28 d. Cardiac tissue constructs composed of h9c2 rat cardiac myoblasts and normal adult human dermal fibroblasts in hydrogel were seeded into devices and exposed for 96 h to 100 mmHg/10 mmHg pressure (0.4 s/0.6 s) at 1 Hz. "Systolic" pressure was generated via pneumatic pump while a hydrostatic pressure head gave rise to "diastolic" pressure. The controls were exposed to “normal” stretch while the experimental group was exposed to “volume overload”.

Results and Conclusions:
The in vitro model yielded similar fibrosis-related gene expression profiles (see figure) and patterns of fibrosis as the murine model.
Abstract P1133: Association Of Mesenchymal Stem Cells Therapy And An Agonist Of Adenosine Receptor On Monocrotaline-induced Pulmonary Arterial Hypertension In Rats
Authors: Dematté Bruno , Da Silva Jaqueline S , Vasques Juliana F , Rocha Bruna , Montagnoli Tadeu , Nascimento-Carlos Bianca , Maia Rodolfo , Barreiro Eliezer , Mendez-Otero Rosalia , Zapata-Sudo Gisele
Published: November 2022

Introduction:
Pulmonary arterial hypertension (PAH) is characterized by an intense inflammatory component leading to vascular remodeling, increasing the pulmonary artery pressure, eventually resulting in right ventricular dysfunction.

Hypothesis:
Association of human mesenchymal stem cells (hMSC) therapy and LASSBio-1860 (agonist of adenosine receptors) would reduce the inflammatory response and vascular remodeling, decreasing the vascular resistance and pulmonary artery pressure, improving right ventricle function and reducing hypertrophy.

Methods:
Protocols were approved by Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. PAH was induced in male Wistar rats by injection of monocrotaline (MCT; 60 mg/kg, i.p.) and confirmed within 14 days by transthoracic echocardiography. Groups were orally treated daily during 14 days with vehicle, LASSBio-1860 (180 μmol/kg) after a single intravenous injection of 10
5
hMSC At the end of protocol, tissues were collected and processed to evaluate expression of proteins using immunohistochemical and western-blotting analysis.

Results:
Mean pulmonary artery acceleration time (PAAT) was reduced in MCT-induced PAH group compared to the control group from 31.0 ± 1.5 to 21.9 ± 1.3 ms. In contrast, the association of LASSBio-1860 and hMSC has recovered the PAAT to 31.0 ± 2.0 ms, which effect was not observed in isolated treatment either with LASSBio-1860 or hMSC (23.5 ± 2.1 and 22.2 ± 2.2 ms). Table 1 shows hemodynamic, molecular and immunohistochemical parameters.

In conclusion, the association of LASSBio-1860 and hMSC improved hemodynamic, morphologic and inflammatory parameters in an MCT-induced model of PAH.
Abstract P1134: Activation Of Endogenous Transcription Factors By CRISPRa Mediates Cardiac Reprogramming
Authors: Keepers Benjamin P , Qian Li , Liu Jiandong , Shutt Samuel
Published: November 2022

Studies of cardiac reprogramming - the conversion of fibroblasts to cardiomyocyte-like cells - have deepened our understanding of cell identity changes, and they hold promise for potential clinical application. At the core of most cardiac reprogramming approaches lie three transcription factors: MEF2C, GATA4, and TBX5 (MGT). Exogenous expression of MGT can convert fibroblasts into cardiomyocyte-like cells, but it is unknown if activation of endogenous MGT can mediate reprogramming, or if these reprogrammed cells would be phenotypically different. In this study, we implemented CRISPR-based gene activation (CRISPRa) for targeted activation of endogenous MGT in mouse and human fibroblasts. Transcriptional activation ranged from 10X-10,000X greater than the non-targeted control, varying by gene and target locus. In combination with miR-133 overexpression, CRISPRa of all three of the MGT factors induced cardiac reprogramming of human fibroblasts. Multiple combinations of guide RNAs were capable of mediating human reprogramming with varying efficiencies. Induced cardiomyocytes appear phenotypically different under immunofluorescence. Finally, we simplified the human CRISPRa platform by combining the guide RNAs into a single, multiplex vector. Cellular and molecular studies of this new reprogramming model may provide mechanistic insights into cell fate conversion.