bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1040: The Protective Effects Of Sialidase Neu3 Against Cardiac Ischemia And Reperfusion Injury
Authors: Piccoli Marco , Lavota Ivana , Coviello Simona , Ghiroldi Andrea , Cirillo Federica , Pappone Carlo , Anastasia Luigi
Published: November 2022

The salvage of myocardium after acute myocardial infarction (AMI) can only be achieved by timely reperfusion. This is life-saving, however it may also lead to additional damage in the form of ischemia and reperfusion injury (IRI). Protecting cardiac tissue from IRI remains one of the greatest unmet clinical needs in cardiology. Therefore, a better understanding of IRI pathophysiology is necessary. There has been increasing recognition that the activation of specific molecular mechanisms, including the Reperfusion Injury Salvage Kinase (RISK) pathway and HIF-1α contributes to cardioprotection against IRI. Along this line, we previously reported that sialidase Neu3, which removes sialic acid from glycosphingolipids, controls HIF-1α activation via a prolyl hydroxylase-independent pathway. In fact, we found that Neu3 was up-regulated under chronic hypoxia in cyanotic congenital cardiac patients and its overexpression increased muscle cells’ resistance to hypoxic stress by activating the RISK pathway. In light of these premises, the goal of this study was to determine Neu3's involvement in the cardiac response to IRI and establish whether its inducible activation could promote cardioprotection. An IRI model in mice, obtained by temporarily blocking the LAD coronary artery, showed that Neu3 is modulated during this process. Notably, Neu3 showed progressive downregulation during the ischemic phase followed by its reactivation during reperfusion. These results were similar to the Neu3 modulation we observed in human cardiac cells (AC16) exposed to an IRI model
in vitro
. The overexpression of Neu3 during ischemia had beneficial effects both in terms of cell proliferation and reduction of oxidative stress after reperfusion. We found that Neu3 promotes the activation of glycolytic enzymes during the ischemic phase as well as the maintenance of the mitochondrial membrane potential, suggesting that the Neu3-overexpressing human cardiomyocyte undergoes more aerobic metabolism even in oxygen-deficient environments. In conclusion, these results support a role for Neu3 in cardioprotection against IRI, suggesting that it may be a useful therapeutic target for improving the recovery of patients who have undergone acute myocardial infarction.
Abstract P1044: A Drug-Like Activity Of Polydopamine Nanoparticles Mediates Cardiomyocyte Bio-Modulation And Maturation
Authors: Baseeruddin Alvi Syed , Sridharan Divya , Shalaan Mahmoud , mergaye muhamad , Khan Mahmood
Published: November 2022

Introduction:
Bio-active nanomaterials are being extensively explored for various biomedical and tissue engineering applications. Polydopamine nanoparticles (PDA-NPs) is a self-assemble polymer derived from dopamine which has demonstrated anti-inflammatory potential by modulating immune cells. The use of PDA-NPs as bio-active nanomaterial for cardiac tissue repair/regeneration warrants further exploration.

Hypothesis:
We hypothesize that the polydopamine nanoparticles could modulate cardiomyocyte activity due to the presence of catechol moieties and may alter the gene expression and promote maturation of cardiomyocyte.

Results:
The PDA-NPs were fabricated by oxidative polymerization of dopamine monomers which resulted in formation of spherical nanoparticles. These nanoparticles were in the range of 100- 200 nm as evident from SEM and TEM imaging. The FTIR analysis of PDA-NPs revealed the characteristic peaks confirming the presence of quinone and catechol groups. These functional groups play important role in surface adhesion, thus imparting adhesive properties to PDA-NPs. The PDA-NPs were exposed to human iPSC-derived cardiomyocytes (HiPSC-CMs) and their real-time activity was monitored by using MEA. An instantaneous change in the beat period of cardiomyocytes was observed in a dose dependent manner. Moreover, a significant shortening in field potential duration was also seen, these finding suggest that the PDA-NPs stimulates the cardiomyocytes through catechol groups thereby exhibiting strong adrenergic effect. These finding demonstrates that PDA-NPs could significantly modulate the contractility of cardiomyocytes and could be used for enhancing cardiac contractility. Furthermore, prolonged incubation of PDA-NPs showed accumulation in the perinuclear region as revealed by fluorescence imaging. The influence of intracellular accumulation of PDA-NPs on gene expression was assessed by qRT-PCR. A significant increase in MYL2/MYL7 ratio, GJA5 and TNNI3 was noted, demonstrating the maturation of cardiomyocytes.

Conclusion:
Overall, the treatment of hiPSC-CMs cardiomyocytes with PDA-NPs shows dual response, primarily by modulating the contractility in a dose dependent manner and secondary by promoting maturation.
Abstract P1049: Determination Of Proteome-wide Post-translational Modifications By Lysine Acetylation In Ischemic Cardiac Cells
Authors: Rajakumar Aishwarya , de Lima Isabela Beatriz , Gupta Manish K
Published: November 2022

Introduction:
In ischemic cardiomyopathy, cardiac muscle gets damaged due to ischemia and the heart loses its ability to pump blood properly, leading to heart failure. Considering the effect of ischemic cardiomyopathy on the global population, it is vital to understand the impact of ischemia on the cardiac cells and how ischemic condition changes different cellular functions through post-translational modifications (PTM) of cellular proteins. Acetylation, one such PTM, is known to affect protein synthesis, epigenetic modification, and metabolism. This study aims to identify global acetylation changes due to ischemic conditions.

Methods:
Rat ventricular cardiomyocytes were exposed to reduced oxygen conditions for 12 hours, and the proteins were isolated for acetylation analysis. Mass spectrometry was performed on these proteins to analyze global changes in acetylation due to ischemia.

Results:
Our result shows that 180 proteins are differentially acetylated during ischemia, and these acetylated proteins are found to be localized in different subcellular organelles like mitochondria, nucleus, cytoplasm, etc. These PTMs tune the cellular protein function and regulate different cellular functions such as protein synthesis, epigenetic modification, and metabolism.

Conclusion:
Understanding cellular changes due to post-translational acetylation modification may help in the design of therapeutic agents against ischemia-mediated heart disease.
Abstract P1055: Co-chaperone Bag5 Protects Cardiomyocytes From Oxidative Stress
Authors: Mukker Avni , Randhawa Puneet , de Lima Isabela Beatriz , Gupta Manish K
Published: November 2022

Introduction:
Cardiovascular disease is one of the leading causes of death globally. Cardiac ischemic can alter cellular homeostasis and potentially lead to heart failure. Ischemic cells modulate the expression of molecular chaperones, which assist with the folding/refolding of cellular proteins. Specifically, the heat-shock protein 70 (Hsp70) chaperone, and its co-chaperones in the Bcl-2-associated athanogene (BAG) protein family, help maintain protein homeostasis by protein folding and degradation. This project serves to explore the role of BAG5 in cardiac ischemia.

Methods:
Expression of BAG5 was determined in left ventricular tissue isolated from cardiomyopathy patients and in ischemic cardiomyocytes. Co-immunoprecipitation (IP) and mass spectrometry analysis were utilized to determine potential interacting partners of BAG5. For the generation of ischemic conditions, neonatal rat cardiomyocytes were incubated in a hypoxia chamber for 12 hours in DMEM media with no glucose, 0% serum, and 0.1% O2. The role of BAG5 in cellular viability, reactive oxygen species (ROS) production, and proteostasis during ischemia was determined through gain-in and knock-down approaches using adenoviruses. In addition, mitochondrial membrane potential and ROS production were determined by the TMRE and MitoSOX red staining, respectively. Mitochondrial function was determined by a Seahorse XF assay.

Results:
Co-immunoprecipitation and mass spectrometry analysis revealed that BAG5 interacts with Myosin and Hsp70. Expression of BAG5 in cardiomyocytes was significantly decreased during ischemia. Further, overexpression of BAG5 protects cardiomyocytes by inhibiting excessive autophagy and reducing ROS production. Moreover, BAG5 expression improves mitochondrial function during ischemia.

Conclusion:
Understanding the role of BAG5 within the cell may help establish the protein as a potential therapeutic molecule for the protection of the ischemic heart.
Abstract P1056: Stretch Reduces Inter-filament Spacing And Triggers Alterations In Thick-filament Structure In Healthy Left-atrial Fibers
Authors: Ma Weikang , Papadaki Maria , Kirk Jonathan A , Rupert Cassady , del Rio Carlos L , Irving Thomas C
Published: November 2022

Introduction:
Left-atrial dilatation is thought to play a key pathological role in facilitating remodeling in both genetic and acquired cardiac myopathies, as well as enabling the onset and maintenance of atrial fibrillation. However, despite its likely role in disease, the structural responses of the atria to stretch remain understudied. The myofilament responses to stretch have been extensively studied in ventricular tissue, where strain-dependent recruitment of reserve, OFF-state, myosin heads from the thick-filament contribute to length dependent activation (LDA, i.e., greater forcer at longer sarcomere lengths). In these experiments, the effects of stretch in both atrial sarcomere and thick-filament structure were evaluated.

Methods:
Myofilament structure was evaluated via x-ray diffraction at two sarcomere lengths (SL, SHORT: 2.1 μm, and LONG: 2.3 μm) in relaxed left-atrial and left-ventricular myocardial skinned fibers from Yucatan mini-pigs. In these experiments, both equatorial and meridional reflections reflecting sarcomeric structure and ordering were evaluated. In addition, the biomechanical effects of stretch were also evaluated in both skinned porcine fibers and engineered heart tissues (EHT) from atrial and ventricular hIPSc-derived myocytes via a proprietary system (MyoPod; Propria Biosciences).

Results:
In both atrial skinned fibers and EHTs, stretch triggered moderate increases in force, demonstrating LDA. LA fibers, stretch decreased lattice spacing but failed to alter the I
11
/I
10
equatorial intensity ratio, suggesting negligible length-dependent recruitment of OFF-state myosin heads from the thick-filament. LA fibers, however, tended to have higher I
11
/I
10
values at short SL (vs. LV fibers) suggesting an intrinsically higher population of ON-state myosin heads. In addition, stretch triggered alterations in thick filament ordering in both LA and LV fibers, as reflected by changes in both the 3
rd
and 6
th
order meridional reflections (M3 and M6), suggesting effects in both myosin heads and thick-filament backbone.

Conclusion:
Atrial stretch results in marked sarcomere structural alterations, reducing both inter-filament spacing and thick-filament ordering that likely contribute to length dependent activation. The role of these alteration in atrial pathophysiology remains to be studied
Abstract P1068: BubR1 Is A Novel Regulator Of Cardiac Development And Conduction
Authors: Pun Renju , Jindra Katelyn , Noss Bryant , Kim Michael , North Brian
Published: November 2022

BubR1 (Budding uninhibited by Benzimidazole Related -1) is a kinase with a well-established role in regulating the spindle assembly checkpoint during mitosis. Recent studies have found that BubR1 insufficiency leads to premature aging phenotypes, and that BubR1 protein levels naturally decline with age, suggesting a role for BubR1 in aging and age-related diseases. Interestingly, mice carrying hypomorphic alleles of BubR1 expressing approximately 10% of the protein (BubR1 hypomorphic mice,

BubR1
H/H

) die suddenly in a manner reminiscent of sudden cardiac death. Subsequent studies further demonstrated that BubR1 hypomorphic mice suffer from conduction abnormalities such as a prolonged QT interval. This abnormality can cause irregular rhythm in the ventricles that can lead to ventricular fibrillation and sudden cardiac death.
However, the molecular and cellular basis linking these phenotypes to deficiency of BubR1 remain unknown.
To define a role for BubR1 in the heart, we have taken a cellular and organismal approach to test the hypothesis that BubR1 regulates cardiac conduction. Utilizing a conditional BubR1 knockout mouse model, we have crossed this mouse to a cardiac-specific cre driver (
Nkx2.5-cre
) to assess the consequence of loss of BubR1 specifically in the heart. We observed that homozygous cardiac-specific deletion of BubR1 is embryonic lethal, indicating a critical role for BubR1 in heart development. Interestingly, electrocardiography (ECG) analysis of heterozygous deletion of BubR1 (55.4 ms ±3.3) also led to a prolonged QT interval
in vivo
as compared to wild type control (46.4 ms ±2.3) (N=10, p < 0.01). To determine the molecular basis for our observed phenotypes
in vivo,
we have performed gene expression profiling on RNA isolated from hearts of 6-month-old

BubR1
H/H

and wild-type mice in which genetic pathways relating to cardiac conduction such as ion channels were altered in expression (N=3). Finally, we have also identified that intercellular communication is dysregulated upon depletion of BubR1.
Together these studies suggests a novel mechanism by which loss of BubR1 may lead to adverse consequences for the heart

in vivo

.
Abstract P1070: Role Of Vezf1 In Cardiomyocyte Development
Authors: Mensah Isaiah K , Tan Hern , Emerson Martin , He Ming , Gowher Humaira
Published: November 2022

Cardiovascular diseases (CVDs) are the leading cause of high morbidity rates and death worldwide. The heart is the first organ that is formed during mammalian development. Progenitor cells that form the cardiovascular system (heart and blood vessels) originate from the mesoderm post gastrulation. To date, there remain considerable gaps in the molecular mechanisms that govern the differentiation of cardiac progenitor cells into a functional heart, knowledge of which will provide advancements in cardiac regenerative medicine. Vascular endothelial zinc finger 1 (Vezf1) is a nuclear protein expressed predominantly in the mesoderm of a developing mouse embryo. A recent study showed that diseased human myocardia had reduced expression of Vezf1 when compared to healthy hearts. However, the function of Vezf1 in heart development is largely uncharacterized and understudied. Using murine embryonic stem cells (mESCs), we sought to investigate the early role(s) of Vezf1 in cardiovascular development. We show that genetic ablation or conditional knockdown of Vezf1 in mESCs impairs cardiomyocyte differentiation, as determined by the absence of contractile activity in these mutants. The absence of cardiomyocytes from Vezf1 mutant cell lines was confirmed using gene expression analysis and immunofluorescence. We observed impaired mesoderm induction in Vezf1 mutants despite external stimulation of Wnt signaling using small molecule inhibitors, suggesting a potential role of Vezf1 in the modulation of genes in the signaling pathway. Indeed, our gene expression analysis and ChIP-qPCR indicate that Vezf1 potentially associates with the promoters of Wnt signaling genes to activate their expression. These studies point towards a model where Vezf1 regulates the formation of cardiac progenitor cells by modulating the Wnt signaling pathway. Our observations suggest that Vezf1 may sit at the center of determining the fate of mesodermal cells into cardiovascular progenitors, the absence of which impairs cardiomyocyte differentiation. This study is the first to delineate the functional role of Vezf1 in the early development of cardiomyocytes and may form the foundation for future cardiac regenerative medicine.
Abstract P1072: Hypoxia Inhibits Cellular Contractility Via Allosteric Regulation Of Microtubule Acetylation
Authors: Deb Roy Abhijit , Gross Evan G , Pillai Gayatri , Seetharaman Shailaja , Etienne-Manneville Sandrine , Inoue Takanari
Published: November 2022

Hypoxia is implicated in pathogenesis of many cardiovascular diseases (CVDs), including loss of cardiac contractility; but its effects on cytoskeletal dynamics are not well understood. The microtubule (MT) network provides mechanical strength to cardiomyocytes and its dysregulation is observed in many CVDs. Acetylated MTs provide structural flexibility to cardiomyocytes and protect against proteinopathy-induced cardiac failure. MT acetylation is exclusively catalyzed by α-TAT1, but little is known about how α-TAT1 is regulated. Here we report that hypoxia inhibits MT acetylation and promotes nuclear accumulation of α-TAT1. We show that cytosolic localization of α-TAT1, disrupted in hypoxia, is critical for MT acetylation. Using computational, live cell microscopy and biochemical approaches, we identified a conserved localization motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements - nuclear export, nuclear import, and cytosolic retention. Inhibiting Exportin 1 induced nuclear accumulation of α-TAT1. We also found that α-TAT1 cytosolic localization is mediated by CDK1, CK2 and PKA kinases, and inhibited by PP2A phosphatase, through a critical residue: T
322
that binds to 14-3-3 proteins. α-TAT1 knock-out (KO) fibroblasts show defects in actomyosin contractility and focal adhesions. We developed an optogenetic tool, named optoATAT1, which rapidly and reversibly shuttled from nucleus to cytosol on blue light stimulation. optoATAT1 stimulation in HeLa cells increased MT acetylation unlike those kept in dark, validating the tool. optoATAT1 stimulation also led to increase in cell contractility indicated by myosin accumulation and focal adhesion maturation, thus confirming a causal relationship between MT acetylation and cell contractility. Also, α-TAT1 KO fibroblasts are highly proliferative, which may pertain to cardiac hypertrophy. In summary, we have identified a novel role of α-TAT1 C-terminus in regulating MT acetylation and cell contractility under hypoxia. Along with this novel regulatory mechanism of MT acetylation, multiple pharmacological agents identified in this study to modulate spatial regulation of α-TAT1 may offer new insights into the treatment of CVDs.
Abstract P1073: Pathways Regulating Cardiac Myocyte Proliferation
Authors: Zhang Zhenhe , Davenport George , Zhang Yiqiang , Maclellan Robb W
Published: November 2022

Introduction:
Several pathways have been shown to promote cardiomyocytes (CMs) proliferation and regeneration including the Hippo signaling pathway. Our lab demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases the proliferation of adult CMs (ACMs). However, all these stimulations are insufficient to promote robust ACM proliferation. The effect of combining these interventions on CM proliferation and myocardial regeneration remains unknown.

Hypothesis:
KDM4D-mediated CM-specific H3K9 demethylation and Hippo pathway inhibition have additive roles in promoting CM proliferation.

Methods and results:
miR-199 overexpression preferentially increased S-phase CMs (EdU
+
) in cultured neonatal rat ventricular cardiomyocytes (NRVMs), while H3K9me3 demethylase KDM4D preferentially increased G2/M markers (phospho-H3). The combination of KDM4D and miR-199 increased total cell number of NRVMs compared to either alone. Since the effects of miR-199 on CM proliferation are mediated through inhibition of Hippo signaling, we inhibited the pathway directly via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1). Similarly, Sav1 inhibition also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducibly activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA-mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. In vitro gene expression analysis demonstrated that KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle while miR-199 and si-Sav1 preferentially upregulated genes involved in G1/S phase. RNA-seq and promoter analysis demonstrated that KDM4D and Hippo signaling pathway regulate CMs proliferation through different transcriptional pathways. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc.

Conclusions:
KDM4D additively induces CMs proliferation with the Hippo-Yap1 pathways. This may have important implications for strategies that target cardiac regeneration in treating heart disease.
Abstract P1074: High-fat High-sugar Diet Promotes Expression Of The Stress Response Protein REDD1 In Cardiomyocytes By Activation Of A PERK/ATF4 Signaling Axis
Authors: Stevens Shaunaci A , Toro Allyson L , Pfleger Jessica M , Bradley Elisa A , Dennis Michael D
Published: November 2022

Obesity and type 2 diabetes represent a surging public health crisis, impacting nearly half of American adults. Heart failure is a leading cause of mortality and morbidity in patients with diabetes and obesity. Much remains unknown regarding the molecular players that contribute to heart failure, but at the cellular level ER stress and inflammation are considered key hallmarks of cardiomyocyte dysfunction. The stress response protein regulated in development and DNA damage 1 (REDD1) is upregulated as a consequence of ER stress in multiple pathological conditions and was recently shown to promote inflammatory signaling. However, a role for diabetes-induced REDD1 in the context of heart failure has yet to be explored. Herein, we investigated the hypothesis that consumption of a pro-diabetogenic diet enhances REDD1 expression in cardiomyocytes by promoting ER stress. C57BL6/N mice were fed either a control chow or a high-fat high-sugar (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 weeks. Mice fed a HFHS diet developed obesity and glucose intolerance. Hearts of mice fed a HFHS diet had an abundance of REDD1 mRNA and exhibited an increase in ER stress and inflammation characterized by phosphorylation of the ER kinase PERK, XBP-1 splicing, and IL-1β expression. We found that exposing both rat H9c2 and human AC16 cardiomyocytes to the saturated fatty acid palmitate and/or hyperglycemic conditions enhanced REDD1 mRNA and protein expression concomitant with an increase in markers of ER stress. PERK inhibition was sufficient to prevent the increase in REDD1 mRNA expression in cardiomyocytes exposed to hyperlipidemic conditions. Similarly, the transcription factor ATF4 was required for the increase in REDD1 mRNA and protein in cells exposed to hyperlipidemic conditions. We also found that REDD1 deletion was sufficient to reduce pro-inflammatory cytokine expression in human cardiomyocytes exposed to hyperglycemic and hyperlipidemic conditions. Overall, the findings provide evidence that HFHS environments contribute to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.
Abstract P1081: Palmitoylation-dependent Regulation Of Rab3gap1, Cardiomyocyte Exocytosis, And Atrial Natriuretic Peptide Secretion
Authors: Essandoh Kobina , Subramani Arasakumar , Brody Matthew
Published: November 2022

Palmitoylation is the reversible post-translational modification of proteins with fatty acids catalyzed by zDHHC S-acyltransferases that functions as a potent regulatory mechanism controlling protein trafficking and signal transduction. To investigate functions of palmitoylation in cardiac homeostasis and pathophysiology, we generated transgenic mice with cardiomyocyte-specific overexpression of the Golgi enzyme, zDHHC9, which was previously reported as the Ras S-acyltransferase and is associated with X-linked intellectual disability. Although we did not observe altered Ras palmitoylation, zDHHC9 transgenic mice develop age-dependent cardiac hypertrophy that progresses to functional decompensation and cardiomyopathy. We found palmitoylation of Rab3 GTPase activating protein 1 (Rab3gap1) was significantly elevated in zDHHC9 overexpressing hearts prior to disease onset concomitant with enhanced GTP-loading of Rab3a, suggesting zDHHC9-mediated palmitoylation impairs Rab3gap1-dependent nucleotide cycling on Rab3a. Consistent with this, zDHHC9 overexpression resulted in enhanced Rab3gap1 retention at the Golgi and expansion of Rab3a-positive vesicles at the cardiomyocyte periphery. Co-immunoprecipitation analysis in HEK293AD cells confirmed that zDHHC9 elevation diminishes interaction between active Rab3a and Rab3gap1. To interrogate roles of zDHHC9 in cardiomyocyte exocytosis, we assessed secretion of atrial natriuretic peptide (ANP) and surprisingly found that while zDHHC9 overexpression increased intracellular retention and reduced release of ANP, knockdown of zDHHC9 promoted phenylephrine-induced ANP secretion. Taken together, these data suggest zDHHC9-mediated palmitoylation of Rab3gap1 represses ANP secretion by disrupting GTP:GDP cycling on Rab3a necessary for release of ANP from secretory granules. These studies reveal unexpected functions for zDHHC9-mediated palmitoylation in modulation of secretory pathway flux and regulated hormone secretion via downstream impacts on Rab3a-mediated vesicle fusion and exocytosis and suggest novel roles for zDHHC9, Rab3gap1, and Rab3a in natriuretic peptide secretion.
Abstract P1082: Activation And Phosphorylation Of Rap1b In The Human Megakaryocytic Leukemia Cell Line DAMI
Authors: Hetcher Joshua , Wisinski Jaclyn A
Published: November 2022

In platelet-producing megakaryocytes, the signaling protein Rap1b mediates both activation of integrins for cell adhesion and activation of the transcription factor ERK leading to gene expression changes. As a small G-protein, Rap1b acts as a molecular switch with an inactive GDP-bound state and an active GTP-bound state. Additionally, the association of Rap1b within the inner side of the plasma membrane is dictated by cAMP-dependent protein kinase (PKA) phosphorylation. In an unphosphorylated state, Rap1b is attached to the plasma membrane via a lipid anchor. Upon phosphorylation, Rap1b detaches from the membrane and is cytosolic. We hypothesize the location of active Rap1b, dictated by the guanine nucleotide bound (GDP/GTP) and its phosphorylation status, determines which signaling pathways Rap1b contributes to in megakaryocytes. To evaluate this hypothesis, we first need to optimize methods to detect Rap1b activation (GTP binding) and phosphorylation. Using the megakaryocyte cell line (DAMI), we over-expressed green fluorescent protein (GFP)-tagged Rap1b and treated with reagents to stimulate activation or phosphorylation and then use pull-down assays with the Rap binding domain (RBD) to capture activated Rap1b or pan phospho S/T antibodies, respectively. In addition, the activation of downstream proteins like ERK were evaluated in DAMI cells overexpressing GFP-tagged Rap1b phosphorylation mutants. Overall, this work seeks to determine how the spatial distribution of Rap1b, dictated by phosphorylation influences cellular processes.
Abstract P1083: Calcium-dependent Proteolysis Of Mitochondrial Opa1 By Calpain Contributes To Arrhythmogenesis In Catecholaminergic Polymorphic Ventricular Tachycardia
Authors: Hamilton Shanna , Terentyeva Radmila , Veress Roland , Perger Fruzsina , Nichtova Zuzana , Martin Benjamin , Gyorke Sandor , Belevych Andriy , Csordas Gyorgy , Terentyev Dmitry
Published: November 2022

Intracellular Ca
2+
cycling in ventricular myocytes (VMs) is tightly linked to mitochondrial metabolic output, and disturbances in this coupling contribute to cardiac arrhythmias. We recently demonstrated in the heritable arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT), the arrhythmogenic phenotype is revealed by oxidation of the cardiac ryanodine receptor (RyR2) calcium release channel by mitochondrial reactive oxygen species (mito-ROS), secondary to causative genetic mutation. However, exact mechanisms linking RyR2 dysfunction to mitochondrial injury remain unclear. We hypothesized that RyR2 hyperactivity activates the intermembrane space (IMS)-residing mitochondrial Ca
2+
-dependent protease , driving proteolysis of OPA1, a protein responsible for cristae flattening. This results in altered mitochondrial cristae diameter and mito-ROS production, exacerbating the arrhythmogenic CPVT phenotype. To test this, we created a unique CPVT RyR2-S2222L
(+/-)
rat model. Electron microscopy studies revealed a significant increase in cristae diameters in Langendorff-perfused CPVT rat hearts. Western blot analysis showed significantly reduced OPA1 expression in isolated CPVT VMs, suggesting that Ca
2+
-dependent proteolysis of OPA1 could underlie these ultrastructural changes. Pilot experiments suggest specific inhibition of via adeno-associated viral injection of IMS-targeted calpastatin (CAST, 10
12
VP, 4 weeks) can reduce ventricular tachycardia incidence of CPVT rats in vivo. Adenoviral expression of IMS-CAST in isolated VMs restored OPA1 expression levels and significantly improved intracellular Ca
2+
cycling in CPVT, suggesting a marked improvement in RyR2 function. Furthermore, genetic probes mtRCamp1h and HyPer-7 revealed that IMS- inhibition significantly improves mitochondrial matrix Ca
2+
homeostasis and mito-ROS in CPVT VMs, respectively. Importantly, these beneficial effects were lost in VMs with shRNA-mediated knockdown of OPA1. Our data suggest that enhanced activity of RyR2 promotes IMS- -mediated degradation of OPA1, resulting in mitochondrial structural damage that contributes to proarrhythmic remodeling in CPVT.
Abstract P1085: Transient Receptor Potential Cation Channel Subfamily M Member 7 Deficiency Prevents Ventricular Tachycardia During Myocardial Ischemia Reperfusion
Authors: Li Xin , Feng Jianlin , Yue Lixia
Published: November 2022

Introduction:
Ventricular arrhythmia caused by myocardial ischemia reperfusion (myo-I/R) remain a substantial challenge worldwide. Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a Ca
2+
-permeable channel, and is involved in cardiogenesis and atrial fibrillation. However, the function of TRPM7 in ventricular arrhythmias during myo-I/R is unknown.

Hypothesis:
We propose that TRPM7 plays a role in ventricular arrhythmias during myo-I/R, therefore deletion of
Trpm7
will protect mice against ventricular arrhythmia during myo-I/R.

Methods:
We used myo-I/R mouse model, in combination with patch-clamp, and various other techniques to investigate the potential role of TRPM7 in myo-I/R injury. Intracardiac programed electrical stimulation was performed to explore the susceptibility to ventricular arrhythmia, and in vitro assays were used for mechanism investigation.

Results:
We found that myo-I/R can readily induce spontaneous ventricular tachycardia (VT) in WT mice, whereas global
Trpm7
deletion (
Trpm7
-/-
) dramatically reduced incidence of VT by 50.0% (
P
<0.05,
n
=10). In the program induced VT,
Trpm7
-/-
drastically shortened the duration of VT by 153.5 ± 24.0 (s) (
P
<0.05, n=10). As TRPM7 is highly expressed in both myocytes and cardiac fibroblast, we established myocyte-specific
Trpm7
deletion and fibroblast-specific
Trpm7
deletion mice. We found that TRPM7 in myocytes is responsible for VT induced by myo-I/R. To understand the mechanism by which TRPM7 is involved in VT, we evaluated electrphysiological properties of myocytes. The action potentials (AP) duration of ventricular myocytes isolated from WT mice, which significantly shortened after treatment of oxygen glucose deprivation, whereas
Trpm7
-/-
restored the AP by prolong the duration without influencing amplitude. Whole-cell patch clamp was performed to record L-type Ca
2+
and K
+
currents with sodium bisulfite treatment. Interestingly,
Trpm7
-/-
notably normalized the density of K
+
current back to the normal level when compared to WT myocytes subjected to oxidative stress.

Conclusions:
Our results indicate that TRPM7 in myocytes is involved in electrical remodeling during myo-I/R. Inhibition of TRPM7 channel function may serve as a potential target for myo-I/R induced ventricular arrhythmias.
Abstract P1089: Short-QT & Long-QT Associated Trpm4 Mutations
Authors: Aistrup Gary L , Pfeiffer Ryan , Goodrow Robert , Cordeiro Jon , Ercan-Sencicek Adife G , Desai Mayuri , Kontaridis Maria
Published: November 2022

Cardiac arrhythmias are responsible for 200-300 thousand deaths/year. Despite considerable research effort, much remains to be elucidated concerning the underlying mechanisms of arrhythmogenesis. Mutations in transient receptor potential melastatin 4 (TRPM4), a widely expressed Ca
2+
-activated nonselective cation channel, have been associated with causing cardiac arrhythmias. However, direct genotype-phenotype correlation of arrhythmogenic TRPM4 mutant variants are often complicated, as this channel is not primary to the cardiac action potential. Here, we assessed the electrophysiological and post-transcriptional molecular properties of a single mutation (R892C)-TRPM4 associated with short QT syndrome (SQTS) and a triple mutation (R250C|A432T|G582S)-TRPM4 associated with long QT syndrome (LQTS), using stably transfected HEK293 cells. Overall, protein expression of the triple mutant was found to be significantly reduced, with increased proteasomal degradation, but enhanced SUMOylation, as compared to either WT or the single R892 mutant TRPM4 channel. Consequently, expression of R250C|A432T|G582S-TRPM4 was significantly lower at the cellular membrane than either R892C- and WT-TRPM4. In contrast, while total expression of TRPM4 was not significantly different between WT and the R892C single mutant, although the R892C exhibited increase aggregation. Patch-clamp cellular electrophysiology experiments indicated that both single and triple TRPM4 mutant channels could be activated by lower Ca
2+
concentrations compared to WT. However, R892C-TRPM4 channels inactivated faster, while R250C|A432T|G582S-TRPM4 channels inactivated much slower compared to WT. These data as obtained in our homologous recombinant overexpression system reveal that while the R892C-TRPM4 mutant variant exhibited normal-to-higher levels of expression and increased Ca
2+
-activation sensitivity, its tendency to aggregate combined with faster inactivation can result in overall loss-of-function compared to WT, correlative with SQTS. Conversely, while the R250C|A432T|G582S-TRPM4 mutant variant exhibited reduced expression and perturbed trafficking, its increased Ca
2+
-activation sensitivity and slow inactivation can result in overall gain-of-function compared to WT, correlative with LQTS.
Abstract P1091: Granulocyte-Macrophage Colony-Stimulating Factor Signaling Pathway And Antiarrhythmic Effect Of Renal Denervation In Chronic Heart Failure
Authors: Hu Wenfeng , Tu Huiyin , Wadman Michael , Li Yulong , Zhang Dongze
Published: November 2022

Renal denervation (RDN) is widely investigated in multiple studies of sympathetically driven cardiovascular diseases. While the therapeutic potential of RDN for ventricular arrhythmia has been reported, the mechanisms responsible for the antiarrhythmic effect of RDN are poorly understood. Our recent study showed that macrophage expansion-induced neuroinflammation in the stellate ganglion (SG) was a critical factor for cardiac sympathetic overactivation and ventricular arrhythmogenesis in chronic heart failure (CHF). This study aims to investigate if and how RDN decreases ventricular arrhythmias through attenuating neuroinflammation in cardiac sympathetic postganglionic (CSP) neurons in CHF. CHF was induced by acute surgical ligation of the left coronary artery in rats. Completed bilateral RDN was achieved by surgically cutting all the visible renal nerves around the renal artery and vein and followed by application of 70% ethanol around the vessels. Using immunofluorescence staining and Western blot analysis, we found that expression of granulocyte-macrophage colony-stimulating factor receptor-α subunit (GM-CSFRα), and Iba1 (an activated macrophage marker) in macrophages from the SG were increased in 16-week CHF rats, compared with age matched sham rats. In the ELISA experiment, we observed high levels of GM-CSF in kidney, serum, and SG in CHF rats, which were markedly reduced at 3 weeks after RDN. CHF-elevated macrophage activation and expression of GM-CSFRα in the SG were decreased by RDN. Flow cytometry data confirmed that CHF-increased percentage of macrophages in the SG was reduced at 3 weeks after RDN. Patch-clamp data found that RDN reduced the N-type Ca
2+
currents in CSP neurons from CHF rats (50.5+3.3 pA/pF in CHF rats vs. 28.6+2.2 pA/pF in CHF+RDN rats, P<0.05). RDN attenuated CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-hour continuous telemetry recording in conscious rats revealed that RDN decreased the incidence and duration of spontaneous ventricular tachyarrhythmias in CHF rats. Based on these data, we confirm that RDN inhibits cardiac sympathetic overactivation and ventricular arrhythmogenesis through GM-CSF-macrophage activation-neuroinflammation-N-type Ca
2+
channel signaling axis in CHF.
Abstract P1095: Analysis Of Pro-arrhythmic Effects Of The Trisulfated Disaccharide Heparin Fragment, A Potential Antiarrhythmic Drug.
Authors: Vasques Enio R
Published: November 2022

Background:
The heparin fragment called trisulfated disaccharide (TD) acts on the sodium-calcium exchanger by removing excess calcium from cytoplasm with antiarrhythmic action demonstrated in animal models in the reversal of supraventricular and ventricular arrhythmia. However, it is known that drugs with antiarrhythmic potential can induce arrhythmic phenomena, limiting and interrupting the study of new potentially antiarrhythmic drugs.

Objective:
To evaluate the arrhythmogenic potential of TD in relation to the stimulation of hERG channels in human cardiomyocytes.

Material and Methods:
Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in culture were plated onto a fibronectin coated 48 well microelectrode array plate at a density of 25,000 cells per well in iCell® maintenance medium. The plate was incubated for 7 days until a stable beating phenotype was achieved, and the cells were ready for drug treatment. They were dosed with 0.2% DMSO vehicle, E-4031 (0.05μM), or cisapride (0.05μM) as controls (arrhythmogenic drugs). They were also dosed with TD at four concentrations in triplicate. The activity of the cells was recorded prior to dosing and at 1 hour using the Axion Biosystems Maestro MEA system. Beat period (s), fast Na+ slope (V/s), fast Na+ amplitude (mV) and field potential duration (FPD) were evaluated.

Results:
E-4031 treatment resulted in a significant increase in beat period length (decrease in beat rate) and a decrease in the Na slope and amplitude. There was a very large increase in the FPD. Cisapride showed a very similar response. Both compounds caused an increase in the ratio of the FPD to the beat period length. When the cardiomyocytes were incubated with the different doses of TD, there was a slight increase in the beat period length (decrease in the beat rate), which was observed regarding all the doses. There was an increase in the FPD, which is consistent with the change in beat rates suggesting that the change is purely related to beating and not indicative of any hERG effect or other responses. The ratio of the FPD to the beat period length was the same. There was a slight decrease in the sodium slope and amplitude, but they were not significant. Therefore, the only significant change observed was the beat rate.

Conclusion:
TD when exposed to human cardiomyocytes did not present an arrhythmogenic potential in relation to the hERG channels, and this is a desirable behavior for an antiarrhythmic drug.
Abstract P1096: Kinase-independent Function Of PI3Kgamma Modulates Calcium Re-uptake By Regulating CamKII Mediated Phospholamban Phosphorylation
Authors: Mohan Maradumane L , Witherow Conner , Papay Robert , Sun Yu , Stenson Kate , Naga Prasad Sathyamangla V
Published: November 2022

Although phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ
-/-
) show increased ventricular rate/contraction, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes from PI3Kγ
-/-
mice show reduced calcium reuptake by sarcoendoplasmic reticulum (SR) following caffeine. Unexpectedly, PI3Kγ
-/-
cardiomyocytes showed significant reduction in phosphorylation of phospholamban (PLN) at Thr17, a key regulator of SR calcium re-uptake. Furthermore, loss in PLN phosphorylation in PI3Kγ
-/-
cardiomyocytes was associated with augmented interaction with SR calcium ATPase (SERCA). Surprisingly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ
inact
) in global PI3Kγ
-/-
mice (PI3Kγ
inact
/PI3Kγ
-/-
) normalized caffeine-induced calcium re-uptake, PLN phosphorylation at Thr17 and decreased PLN-SERCA interaction. These data suggested kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Since phosphorylation of Thr17 of PLN is carried out by Ca
2+
/Calmodulin dependent protein kinase (CamKII) we probed for the role of PI3Kγ in regulation of CamKII in PLN phosphorylation. Mechanistically, PI3Kγ exhibits scaffolding function in recruitment of CamKII to PLN at SR to mediate PLN phosphorylation. The study unravels a yet to be recognized kinase-independent role of PI3Kγ in regulating PLN with implications in cardiac function.
Abstract P1099: Evidence Of Sialylation Pathway Alterations In Peripheral Blood Of Brugada Syndrome Patients
Authors: Ghiroldi Andrea , Ciconte Giuseppe , Creo Pasquale , Tarantino Adriana , D'Imperio Sara , Piccoli Marco , Cirillo Federica , Micaglio Emanuele , Michelle Monasky , Locati Emanuela , Vicedomini Gabriele , Pappone Carlo , Anastasia Luigi
Published: November 2022

Brugada syndrome (BrS) is a cardiac arrhythmia associated with a higher risk of SCD. BrS is considered a genetic disorder, and the most commonly mutated gene is SCN5A, encoding the alpha subunit of the voltage-gated cardiac sodium channel (NaV1.5). Mutations of SCN5A usually lead to impairment of NaV1.5 functionality, which is considered the main mechanism of the disease. However, SCN5A mutations are responsible for only 30% of BrS cases. Therefore, it is conceivable that other mechanisms such as post-translational modifications (PTMs) affect NaV1.5 activity. Among others, sialylation may alter ion channel activity by carrying a sugar with a negative charge. Alterations in sialylation have been described in several cardiovascular diseases, as myocardial infarction, Chagas disease, and congenital disorders of glycosylation. For these reasons, the aim was to investigate changes in sialylation in BrS patients to obtain new information on the pathogenesis of BrS. Peripheral blood mononuclear cells (PBMCs) were collected from BrS patients and healthy controls. SNA lectin, a sialic acid-binding protein, was used to characterize the protein sialylation status of PBMCs by Western blot and flow cytometry. Gene expression of enzymes involved in the sialic acid pathway was also examined. The results showed a significant decrease in intracellular and extracellular protein sialylation levels in BrS PBMCs compared with controls. In addition, changes in gene expression of enzymes involved in the sialic acid pathway were detected. Moreover, these changes correlated with clinical parameters associated with phenotypic expression of the disease, such as arrhythmogenic BrS substrate area and potential duration. These findings support that BrS should be considered a systemic disease and are consistent with the presence of overlapping non-cardiac pathologies, as epilepsy, cancer, diabetes, skeletal muscle channelopathies, and laminopathies in BrS patients. Moreover, the discovery that molecular alterations can be found in the peripheral blood of BrS patients supports the existence of a biomarker of the disease. It is a challenge to develop an effective diagnostic test to screen broadly for BrS. In this direction, a multiomic study is ongoing in our center.
Abstract P1101: Mitochondrial Ca Uniporter Complex, ROS And CaT Alternans
Authors: Oropeza-Almazan Yuriana , Blatter Lothar A
Published: November 2022

Background:
Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternation in CaT amplitude and is causally linked to atrial fibrillation (AF). Inhibition of mitochondrial Ca uptake via the mitochondrial Ca uniporter complex (MCUC) facilitates CaT alternans and can lead to an increase in mitochondrial reactive oxygen species(ROS) production that compromises the cellular antioxidative capacity. The objective was to determine the mechanisms through which mitochondrial Ca uptake and ROS production determine the susceptibility of atrial cells to develop CaT alternans.

Methods:
CaT alternans was measured in single rabbit left atrial myocytes with fluorescence microscopy using the fluorescent Ca indicator Cal-520/AM, and for simultaneous measurements of ROS and alternans, cells were loaded with CM-DCFDA and Cal-590/AM. CaT alternans was induced by increasing the pacing frequency until stable CaT alternans was observed. The degree of alternans was quantified as the alternans ratio (AR = 1 – S/L, where S/L is the ratio of the small to the large amplitude of a pair of alternating CaTs).

Results:
Exposure of LA myocytes to the specific MCUC inhibitor Ru360 increased the AR by 86%. However, the subsequent stimulation of mitochondrial Ca uptake with the MCUC activator spermine (Spm) decreased AR by 79% (p=0.028). Quercetin (a O2

.-

scavenger) and Tempol (a superoxide dismutase mimetic) lowered CaT AR by 96% (p=0.0007) and 79% (p=0.0029), respectively. An oxidative challenge with tert-butylhydroperoxide (tBHP) increased AR by 140% (p=<0.0001), and the reducing agent dithiothreitol (DTT) decreased AR by 39% (p=0.0194). Moreover, DTT treatment rescued CaT alternans previously enhanced by tBHP. During CaT alternans the rate of ROS production increased by 23% (p=0.026), however Spm treatment attenuated the increased ROS production.

Conclusion:
An oxidizing environment facilitates the development of CaT alternans. During CaT alternans mitochondrial ROS formation is increased and involves MCUC activity.
Abstract P1102: Rna-seq Analyses Reveal Roles Of The Hvcn1 Proton Channel In Cardiac Ph Homeostasis
Authors: Wu Xin , Li Yawei , Maienschein-Cline Mark , Feferman Leonid , Wu Longjun , Hong Liang
Published: November 2022

Introduction:
The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel family. Hv1 channel controls acid extrusion and regulates pH homeostasis in various cell types. Recent evidence showed that the Hv1 were expressed in the heart.

Hypothesis:
We propose the Hv1 channel is associated with cardiac function.

Methods:
To investigate the role of Hv1 in cardiac myocytes, we performed a transcriptomic analysis of wild-type (WT) and Hv1-/- mouse hearts.

Results:
Hv1 null hearts exhibited a differential transcriptome profile compared with wild-type hearts. The RNA-seq data indicating impaired pH homeostasis in Hv1 null hearts were the downregulated NADPH oxidoreductases (NOXs) and decreased expression of Cl-/HCO3- exchanger. Additionally, Hv1 null hearts exhibited differential expression of cardiac ion channels, suggesting a potential role of Hv1 in cardiac electrophysiological remodeling.

Conclusions:
The data indicated that Hv1 was involved in cardiac pH homeostasis and showed that Hv1 was a regulator of gene transcriptional networks controlling NOX signaling and CO2 homeostasis in the heart. The study highlights the importance of Hv1 in cardiac function and may present a new target associated with heart diseases.