bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3131: SIRTUIN5 Modulates Na
+
/Ca
2+
Handling Through Oxidative Stress Dependent Mechanism In Mouse Heart
Authors: Yoon Jin-Young , Chen Biyi , Ling Sheng Song , Lombard David B , London Barry
Published: October 2023

Mutations in cardiac sodium channel Na
V
1.5 (encoded by SCN5A) can cause inherited or acquired arrhythmias, including ~20% of cases of Brugada Syndrome (BrS) and inherited conduction disease. The peak Na
V
1.5 current (I
Na, Peak
) and the late current (I
Na, Late
) govern the fast upstroke and plateau of the cardiac action potential, respectively. Ca
2+
ions impact excitation-contraction coupling, and imbalance in Na
+
/Ca
2+
homeostasis can cause electrical disturbance. BrS is characterized by a right bundle branch block pattern on EKG, arrhythmias, and sudden cardiac death. Recently, potential mutations in SIRT5, including P114T-SIRT5, were identified in small families with BrS. SIRT5 is a member of the Sirtuin family, acts as NAD
+
-dependent deACYLase.We explored the potential role of SIRT5 in BrS using heterologous expression systems and homozygous P114T-SIRT5 knock-in (P114T-KI) mice. SIRT5 co-immunoprecipitates with Na
V
1.5 in HEK293 cells. Live-cell staining using DCFDA or mitoSOX showed that P114T-KI hearts had increased basal reactive oxygen species (ROS) and were more sensitive to oxidative stress induced by H
2
O
2
than wild-type littermates. P114T mice displayed Ca
2+
-mishandling as measured by Ca
2+
imaging in perfused hearts, increased I
Na, Late
, prolonged QTc, and arrhythmias. Notably, treatment with mitochondrial ROS scavenger mitotempo mitigated the aberrant Ca
2+
handling and arrhythmia in P114T mice.Together, these suggest that P114T-SIRT5 causes Na
+
/Ca
2+
mishandling-induced arrhythmias in ROS-dependent manner, highlighting potential mechanisms underlying BrS. They pave the way for using SIRT5 or its activators as novel anti-arrhythmic therapies.
Abstract P3132: Tgfβ Signaling Triggers Nonmyocyte Gene Expression In Tbx18-induced Pacemaker Myocytes And Abbreviates Their Automaticity
Authors: Fan Jinqi , Cho Hee Cheol
Published: October 2023

Background:
We have previously demonstrated reprogramming of ventricular myocytes into induced pacemaker cells (iPMs) using an embryonic transcription factor TBX18. We have also discovered that Tgfβ pathway is significantly upregulated in TBX18-iPMs, and triggers fibrosis upon gene transfer in vitro and in vivo. Here, we sought to investigate the impact of Tgfβ signaling on iPMs’ automaticity.

Methods:
Neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno-TBX18 or control Adeno-GFP. Single cell RNAseq was performed at days 3, 6, and 14. Spontaneous field potentials from NRVM monolayers were recorded with microelectrode array.

Results:
TBX18 gene transfer strongly repressed working cardiomyocyte gene expression, e.g., Gja1, Scn5a, Pln throughout the 14-day period. TBX18-NRVMs progressed toward pacemaker cells, evidenced by increase in nodal pacemaker-related ion channel gene expression. TBX18 also triggered cytoskeletal remodeling, and activated nonmyocyte gene expression throughout the 2-week window. This was illustrated by ECM remodeling gene expression such as Acta2, Col1a1, and Postn. Treatment with A83-01, a small molecule inhibitor of Tgfβ receptors, mitigated the increase in nonmyocyte gene expression. TBX18-NRVMs exhibited spontaneously oscillating field potentials, reaching a peak pacing frequency of 196±36 bpm during week 1, which is comparable to the sinus rhythm of newborn rats. However, the pacing frequency gradually wavered after week 1. In contrast, A83-01-treated TBX18-iPMs showed peak pacing frequency beyond week 1, with the majority of their total beat counts near their sinus rhythm (63.3%) while only 14.9% of total beats were near the sinus rhythm in TBX18-iPMs without Tgfβ inhibition (P<0.001). Interestingly, A83-01 did not impact PM-related ion channel gene expression (such as Hcn4, Gjc1) nor the percentage of TBX18-iPMs (5.1% vs. 5.4%, with or without A83-01, p=0.561), suggesting that improved automaticity may be due to its impact on nonmyocytes.

Conclusion:
TBX18-iPMs exhibit increased Tgfβ signaling and nonmyocyte gene expression, which negatively impacts their automaticity. Inhibition of Tgfβ signaling modulates nonmyocyte gene expression and enhances automaticity of TBX18-iPMs.
Abstract P3135:
In Vivo
Dissection Of CaMKII-Mediated Arrhythmias In CPVT
Authors: Pavlaki Nikoleta , de la Serna Buzon Sofia , Sweat Mason , Pu William T , Marx Steven , Bezzerides Vassilios J
Published: October 2023

Background:
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome of the structurally intact heart manifesting life-threatening bursts of ventricular tachycardias due to dominant gain-of-function mutations in the ryanodine receptor type 2 (RyR2) and β-adrenergic (β-AR) stimulation. Both protein kinase A (PKA) and Ca
2+
- calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR2 channels, but only the latter can initiate the arrhythmogenic phenotype in CPVT. Whether through L-type Ca
2+
channel (LTCC)-mediated Ca
2+
increase or the Epac2-nitric oxide pathway, the exact mechanism by which β-AR-mediated CaMKII activation unmasks lethal arrhythmias in CPVT remains unresolved.

Aim:
To gain mechanistic insight and ablate ventricular arrhythmias in a mouse model of CPVT by selectively disrupting integral components of β-AR signaling.

Methods:
By utilizing either direct adeno-associated virus (AAV) delivery or the CRISPR/Cas9-AAV-based somatic mutagenesis (CASAAV) system, we selectively expressed a mutant LTCC β
2
-subunit, ablated the small GTPase Rad, or disrupted other components of the β-AR pathway in the hearts of mice with the pathogenic RyR2 mutation R4650I as a model of CPVT. The effects of disruption of LTCC regulation were determined by high-speed Ca
2+
imaging of isolated ventricular cardiomyocytes or whole animal electrophysiological (EP) testing for ventricular arrhythmia incidence
in vivo
.

Results & Conclusions:
Our data demonstrate that abnormal RyR2-mediated calcium release events are induced by hyperactivated LTCCs
in vitro
, even in the absence of adrenergic stimulation, with increased arrhythmia incidence
in vivo
. These results suggest that β-AR stimulation leads to pathogenic RyR2 Ca
2+
leak through LTCC-mediated contribution to CaMKII hyperactivity. Alternative mechanisms of CaMKII activation that mediate arrhythmia induction in CPVT require additional future investigation.
Abstract P3137: The C-terminus Of Rad Is Required For Regulation Of The Cardiac Ca
V
1.2 Channel
Authors: Elmore Garrett , Ahern Brooke M , Sebastian Andrea , Lohano Sarisha , McVay Nicholas , Levitan Bryana , Dupont Alec , Andres Doug A , Satin Jonathan
Published: October 2023

Background:
Heart failure with reduced ejection fraction (HFrEF) is a leading cause of hospitalization and death. Current therapies fail to address loss of contractile force. Targeting dysfunctional Ca
2+
handling proteins involved in excitation-contraction, such as Ca
v
1.2 may offer a means to improve quality of life and attenuate adverse cardiac remodeling. RAD is a key target bridging sympathetic modulation of Ca
v
1.2 function. During stress, the beta-adrenergic (β-AR) effector, protein kinase A (PKA), phosphorylates RAD, modulating Ca
v
1.2 current (I
Ca,L
) and increasing cardiac contraction. RAD inhibits current, whereas Rad-knockout phenocopies a β-AR/PKA phosphorylated modulated current. Across RGK family members the highly conserved C-terminus is necessary for membrane association and block of I
Ca,L
in immortalized cell lines. The RAD C-terminus significance is unknown in adult mammalian cardiomyocytes.

Hypothesis:
RAD lacking a C-terminus will be unable to localize to surface membrane Ca
v
1.2 channels; thus Ca
v
1.2 activity will be modulated.

Methods and Results:
A 3xFLAG epitope was added to the N-terminus of the endogenous mouse
Rrad
gene. Full-length FLAG-RAD mice were compared to mice with truncated RAD (A277X). Ventricular cardiomyocytes were isolated for immunocytochemistry and
ex
vivo electrophysiology. Confocal microscopy was used to visualize localization of FLAG-tagged RAD. Full-length FLAG-RAD showed a repeating t-tubular pattern that co-localizes with Ca
v
1.2 staining whereas FLAG-RAD A277X failed to show this pattern. A277X I
Ca,L
showed significantly hyperpolarized activation midpoint (-17 mV) and 2-fold increase in maximal conductance. Additionally, current decay was faster in A277X showing a significant 2-fold decrease in the current 30 ms after peak current.

Conclusions:
Myocardial Ca
v
1.2 channels in a RAD C-terminal deletion model phenocopy Ca
v
1.2 function modulated in response to β-AR stimulation. Mechanistically, the polybasic RAD C-terminus confers Ca
v
1.2 regulation via membrane association of RAD. Interfering with RAD at the C-terminus residues constitute a specific target for boosting heart function as a treatment for HFrEF.
Abstract P3138: Potentiation Mechanism Of SR-Ca2+ Uptake During Myocardial Infarction; A Molecular Explanation Of Ca2+ Arrhythmogenicity Of Purkinje Fibers In The Ischemic Heart.
Authors: Amin Ruhul , Wiede Louisa , Dun Wen , Haq Kazi T , Dore Jules , Ter Keurs Henk E , Boyden Penelope A , Stuyvers Bruno D
Published: October 2023

Experimental and clinical evidence attributes lethal VTs and VFs associated with ischemic myocardial infarctions (
MI
) to abnormal electric signaling in the Purkinje network. In cardiac Purkinje cells (
Pcells
), spontaneous Ca
2+
releases from the sarcoplasmic reticulum (
SR-CaR
) generate depolarizing Ca
2+
waves which, in turn, can mediate DADs and non-driven APs. Increase of SR-CaR was reported in Pcells of dog heart with 48Hrs MI. An augmentation of SR-Ca
2+
uptake (
SR-CaU
) accelerates SR-Ca
2+
recycling and is a possible cause of larger (pro-arrhythmic) SR-CaR in Pcells of ischemic heart. We tested this hypothesis in canine and porcine models of acute MI.Pcells and heart tissues were prepared 48Hrs after LAD coronary artery ligation. Intracellular Ca
2+
activity was captured by confocal microscopy. SR-CaU was studied by analyzing Purkinje-typical Ca
2+
events, namely Ca
2+
-sparks, peripheral Ca
2+
-wavelets and cell-wide Ca
2+
-waves (
CWW
s). Relevance of our analysis was tested with a Purkinje-specific model of Ca
2+
mobilization. Protein/gene expressions of SR-Ca
2+
pump isoforms were studied by immunofluorescence, WBs, and qPCR in dog and pig infarcted tissues.We found that (1) individual Ca
2+
release sites in MI Pcells fired sparks at 61% larger frequency, (2) the duration of wavelet Ca
2+
decay was reduced by 25% due to 37% acceleration of cytosolic Ca
2+
removal (time constant: 156±21ms (Normal; n=10)
vs
98±9ms (MI; n=16; P<0.01)), (3) wavelet amplitude was increased by 50%, and (4) larger waves (CWWs) showed similar alterations. Numerical replication of MI wavelets succeeded with 52% increase of Ca
2+
uptake rate in the model of normal wavelets. Our results are consistent with an augmentation of SR-CaU taking place in Pcells shortly after the onset of MI. Molecular analyses done in the same MI model in pig showed that post-MI increase of SR-CaU results from the substitution of cardiac isoform SERCA2a by the ubiquitous and more potent SERCA2b in the infarcted cardiac tissues including Purkinje fibers. Our study reveals, for the first time, the molecular foundation of Purkinje arrhythmogenicity in ischemic heart and highlights novel targets for diagnosis, prevention, and treatment of risk of sudden cardiac death associated with ischemic MIs.
Abstract P3146: Cardiac Stress-induced Arrhythmias And Myocardial Infarction Via Specific Genetically Phosphodeleted Sites In Connexin 43 Channels
Authors: Munoz Manuel F , Nguyen Thao , Nuno Janet , Contreras Jorge
Published: October 2023

Connexin 43 (Cx43) gap junction remodeling is observed in various cardiac pathologies and is linked with arrhythmogenesis, myocardial infarction, and sudden death. Here, we aim to characterize the contribution of Cx43 phosphorylation toward mislocalization at the intercalated disc. For this, we used two genetically phospho-deleted Cx43 knock-in mouse lines with serine residues replaced by alanine in the corresponding sites for phosphorylation via mitogen-activated protein kinase (MAPK) (S255/262/279/282A; Cx43MK4) or Ca
2+/
calmodulin protein kinase II (CaMKII) (S325A/S328A/S330A; Cx43S3A). Ca
2+
signaling, cardiac electrical signals, and arrhythmias were assessed via optical mapping and telemetry in wild type, Cx43MK4, and Cx43S3A mice. Upon basal conditions or isoproterenol stimulation, wild-type animals did not display arrhythmogenic behaviors. However, after isoproterenol treatment, Cx43S3A mice showed increased QTc and deadly arrhythmias compared to wild-type animals. Isoproterenol challenge only increased the number of single PVCs in Cx43MK4 mice when compared to wild-type. Isoproterenol also evoked myocardial infarction in Cx43S3A hearts, which is negligible in Cx43MK4 and wild-type mice. Interestingly, GAP19, a Cx43 hemichannel blocker, reduced lethal arrhythmias and myocardial infarction in Cx43S3A mice. Isoproterenol challenge induced an increase in arrhythmogenic Ca
2+
events in both Cx43S3A and Cx43MK4 hearts compared to wild-type mice. However, Cx43S3A also displayed prolonged Ca
2+
decay transients that were reduced in the presence of Gap19. Our results suggest that the absence of Cx43 phosphorylation by CaMKII leads to lateralization and opening of Cx43 hemichannels, and consequently severe arrhythmias and myocardial infarction. On the other hand, the absence of MAPK phosphorylation promotes mainly a reduction of Cx43 levels at the intercalated disc region that is only associated with mild arrhythmias.
Abstract P3148: Wnt/β-catenin Signaling Inhibits T-type Calcium Current And Causes Sinoatrial Node Dysfunction
Authors: Florczak Kaya , Lu Aizhu , Wang Jerry , Gu Ruonan , Xia Ying , Wang Hongwei , Davis Darryl R , Liang Wenbin
Published: October 2023

Background:
The T-type Ca
2+
channels (I
Ca,T
) are expressed in the fetal and neonatal cardiomyocytes, but in the adult heart they are exclusively expressed in the cardiac conduction system, including the sinoatrial node (SAN). In contrast, the L-type Ca
2+
channels (I
Ca,L
) are expressed ubiquitously in the adult heart and are important for cardiac contraction. The Wnt/β-catenin signaling (Wnt signaling) is activated in various heart disease and we and others have recently demonstrated that Wnt signaling inhibits cardiac Na
+
current. In the present study, we investigated if calcium channels are also regulated by Wnt signaling.

Methods and Results:
Because neonatal rat ventricular myocytes (NRVMs) express both I
Ca,L
and I
Ca,T
, we investigated the effect of Wnt signaling on NRVMs by treatment with either Wnt3a protein or CHIR-99021 (CHIR) (two different activators of the Wnt signaling). PCR array and RNA sequencing studies showed that Wnt signaling did not affect the mRNA of
Cacna1c
(encoding the α subunit of I
Ca,L
, Ca
v
1.2), but the mRNA of
Cacna1g
(encoding the α subunit of I
Ca,T
, Ca
v
3.1) was reduced in NRVMs (p<0.05, n=3) after treatment with either Wnt3a or CHIR. These observations were verified by qRT-PCR which showed dose-dependent reductions (p<0.05, n=4) in
Cacna1g
mRNA after CHIR treatment. Western blot and patch-clamp also demonstrated reductions (p<0.01, n=5) in channel protein and ionic current density of T-type, but not L-type, Ca
2+
channels after treatment with Wnt3a or CHIR. These observations suggest that Wnt signaling selectively inhibits T-type Ca
2+
channels in cardiomyocytes. Consistent with a critical role of T-type Ca
2+
channels in the pacemaker activity of SAN (the primary pacemaker of the heart), the spontaneous firing rates of isolated rat and mouse SAN tissues were reduced by ML-218 (an I
Ca,T
blocker) in a dose-dependent manner (p<0.01, n=5). In addition, activation of Wnt signaling in adult rat SAN tissues by CHIR led to slower and irregular beating, indicative of SAN dysfunction (p<0.01, n=5).

Conclusions:
These observations are consistent with the conclusions that Wnt/β-catenin signaling selectively inhibits T-type calcium current and may play a role in sinoatrial node dysfunction.
Abstract P3149: Effect Of SGLT2 Inhibitors On Atrial Fibrillation Susceptibility In Diabetic Mice
Authors: Granger Jonathan , Nguyen Pauline , Mesubi Olurotimi O
Published: October 2023

Background:
Diabetes mellitus (DM) and heart failure (HF) are independent risk factors for atrial fibrillation (AF), the commonest clinical arrhythmia. A new class of drugs - sodium glucose cotransporter 2 inhibitors (SGLT2i) have beneficial effect in DM and HF and are now part of the guideline directed therapy for HF. However, conflicting evidence exists regarding their role and effect on cardiac arrhythmias including AF independent of their effect on HF and their exact mechanism(s) of action on the heart.

Objective:
Here we tested the effect of two commonly used SGLT2i on AF susceptibility in a type 2 DM (T2D) mouse model.

Methods and Results:
We used a T2D mouse model (high fat diet + low dose streptozocin) and a cohort of T2D mice were treated with SGLT2i - Empagliflozin (30mg/kg/d) or Dapagliflozin (5mg/kg/d) for 10 weeks in drinking water. We performed invasive electrophysiologic studies (EPS) to assess AF susceptibility by a rapid atrial burst pacing protocol in male mice aged 5 months at the time of EPS. T2D mice had increased AF susceptibility compared with non-diabetic littermate controls (normal chow diet + citrate buffer) [78% (14/18) vs. 28% (5/18), p < 0.01]. T2D mice treated with Empagliflozin were protected from increased AF susceptibility (22% (3/18) [p < 0.01]) while T2D mice treated with Dapagliflozin were not protected from increased AF susceptibility (58% (11/19) [p = 0.29]). Both Empagliflozin and Dapagliflozin treated T2D mice had normalization of their blood glucose levels to comparable levels with the non-diabetic control mice. T2D mice had mild LV dysfunction compared with non-diabetic controls (EF - 66% vs 81%, p < 0.01) and treatment with either Empagliflozin or Dapagliflozin attenuated the degree of LV dysfunction (EF - 71% in both groups).

Conclusion:
Taken together, our data support the concept that SGLT2i decrease AF susceptibility in hyperglycemia primed AF, in addition to attenuating LV dysfunction in T2D. However, the effect on AF was only observed with Empagliflozin but not Dapagliflozin treatment independent of their effect on hyperglycemia and LV dysfunction. Further investigations are necessary to determine if there is a class effect of SGLT2i on AF susceptibility and the specific mechanisms of action modulating this effect.
Abstract P3163: Exosomes From Pseudomonas Aeruginosa Infected Macrophages Drives Cardiomyocyte Contractile Dysfunction
Authors: Kumar Naresh , Ahumada Omar S , Ellendula Maneeth P , Planner Clara , Khan Mahmood , Singh Harpreet , Mahajan Sahil , Lafuse William , Wozniak Daniel , Ganesan Latha , Rajaram Murugesan V
Published: October 2023

Rationale:
Pseudomonas aeruginosa
(
P.a.
) infection is very common in ICU patients and is known to increase the mortality rate. Our lab demonstrated that
P.a.
infection caused severe cardiac dysfunction without disseminating into the heart. However, the mechanism of cardiac complication during
P. a.
infection is not known; understanding this is critical for the development of host-protective therapy. Thus,
we hypothesize that cardiac dysfunction caused by P.a. is either mediated through systemic inflammation and/or the release of exosomes and bacterial outer membrane vesicles from infected cells.

Objective:
Identify the mechanism of cardiac inflammation and cardiac dysfunction caused by
P.a.
infection.

Methods and Results:
To investigate whether
P.a.
infection causes cardiomyocyte contractile dysfunction, we exposed the hiPSC-CMs with
P.a.
and monitored the hiPSC-CMs electrical activity using Multi-electrode array (MEA). We found that the contraction of hiPSC-CMs stopped completely at 8h post-infection. Next, to test whether the inflammatory cytokines, exosomes, and bacterial OMVs released upon infection affect contractile function, we exposed the hiPSC-CMs with conditioned media harvested from
P.a.
infected hMDMs. Our data revealed that exposure of conditioned media to hiSPC-CMs caused severe contractile dysfunction and arrhythmia. Also, we found that conditioned media contains an increased level of inflammatory cytokines IL-1β, TNFα, and IL-6 along with exosomes and OMVs. To test whether heat inactivation of conditioned media preserves the hiPSC-CMs, we inactivated the media and stimulated the cells. Our data suggest that the heat-inactivated conditioned media impaired the hiPSC-CMs contractility, due to the release of exosomal content. Furthermore, based on LC-MS/MS analysis of purified exosomes from
P.a.
infected hMDMs, we identified that exosomes are packed with bacterial toxins and immunogenic antigens.

Conclusion:
Our data demonstrate that
P.a.
releases OMVs and bacterial toxins into the host cells, which are packed in exosomes. Though, infected macrophages release inflammatory cytokines, the main source for hiPSC-CM contractile dysfunction is the release of exosomes packed with bacterial toxins and antigens.
Abstract P3165: Spectrin-based Regulation Of Exosome Secretion In Cardiac Fibroblasts
Authors: Shaheen Rebecca J , Thomas Evelyn , Nassal Drew , Patel Nehal J , xu Xianyao , Hund Thomas J
Published: October 2023

Cardiac fibroblasts (CFs) not only modulate the extracellular matrix under physiological conditions, but also have the ability to sense local injury and respond with a phenotypic change and increased paracrine communication to distal cells. While a myriad of studies have focused on identifying the paracrine signals involved in recruitment and activation of CFs, the manner by which CF paracrine communication networks are tuned in response to chronic stress remains unclear. Previously, our lab found that stress-induced loss of the cytoskeletal protein, β
IV
-spectrin, regulates the CF phenotype and fibrosis. Therefore, we hypothesized that degradation of β
IV
-spectrin is a critical step in stress-induced communication involved in recruitment of distal CFs. In this study, we first collected conditioned media from WT and β
IV
-spectrin deficient (qv
4J
) CFs to test the effects on quiescent WT CFs. We found that the CFs treated with qv
4J
-conditioned media showed increased proliferation and compaction rates, as well as altered gene expression in the recipient cells compared to those treated with WT-conditioned media. To further explore the role of β
IV
-spectrin in paracrine signaling, we isolated exosomes from WT- and qv
4J
-conditioned media. We found that loss of β
IV
-spectrin in CFs resulted in an increased concentration of exosomes in conditioned media, that also induced increased proliferation in quiescent CFs. Furthermore, we found that genetic mice expressing degradation-resistant β
IV
-spectrin (

qv
3J

mice) showed impaired CF recruitment and activation following myocardial infarction (MI) together with increased mortality and impaired fibrotic remodeling compared to WT. Finally, we demonstrate that exosomes from a WT-MI heart can improve fibrotic remodeling in

qv
3J

mice following MI. Our work identifies a novel role of the spectrin-based pathway in facilitating long-range communication in response to chronic stress.
Abstract P3170: Immunomodulatory Effects Of Exosomes From Epigenetic Drugs-Stimulated Macrophages
Authors: Perales Selene , Sigamani Vinoth , Rajasingh Sheeja , Johnson Rajasingh
Published: October 2023

Background:
Exosomes have significant therapeutic promise; however, a key problem prohibiting and exploring exosomes in clinical therapy is the availability of disease-specific exosomes. Our recent in vitro and in vivo studies identified that combined treatment with Procainamide (P, DNMT1 inhibitor) and Tubastatin A (T, HDAC6 inhibitor) reduces inflammation upon lipopolysaccharide (LPS) stimuli. Here, we elucidate the therapeutic potential of exosomes derived from the macrophages treated with P+T during the inflammatory condition.

Hypothesis:
We hypothesize that exosomes derived from LPS-challenged macrophages (THP1) treated with P+T will provide a better anti-inflammatory response than direct drug treatment.

Methods and Results:
Macrophages were grown in exosome-depleted media for 72 hrs and stimulated once with LPS, P+T, or L+P+T for exosome collection. These exosomes were used for treating LPS-challenged macrophages. qRT-PCR analysis was performed to check for the expression of pro-inflammatory genes after exosome treatment. Our results show that LPS-challenged macrophages treated with exosome from L+P+T showed significantly reduced expression of TNFα and IL1β when compared to the direct drug treatments
(Fig. A, B)
. We also observed that the pro-inflammatory microRNAs such as miR-21 and miR181-a were significantly reduced in the L+P+T stimulated exosome group
(Fig. C, D)
.

Conclusions:
We show for the first time that drug-stimulated exosomes are more effective in reducing inflammation than direct drug treatment. These specific exosomes could be used for infection-related therapy.
Abstract P3171: Tubular Mitochondria Dysfunction Aki Induces Cardiac Malfunction In Cardiorenal Syndrome Type 3
Authors: Lin Hugo Y
Published: October 2023

Cardiorenal syndrome type 3 (CRS 3) occurs when an acute kidney injury (AKI) leads to the development of cardiac dysfunction. It is associated with significant morbidity and mortality and is becoming increasingly prevalent. Mitochondrial dysfunction in renal tubular cells has been proposed as a critical regulator of AKI, but the exact mechanism of renal tubular mitochondrial dysfunction AKI-induced CRS 3 is still unclear. This study aims to characterize kidney and heart tissue injuries after AKI induced by maleic acid (MA). In an in vivo model, MA was used to induce AKI in C57BL/6 mice, and in an in vitro model, human proximal tubular epithelial cells (HK-2) were treated with MA. Our results showed that MA-induced AKI exacerbated renal tubular injury, increased the expression of renal injury molecule-1 (KIM-1), and caused apoptosis of renal tubular cells. At the cellular level, mitochondrial dysfunction was associated with increased mitochondrial reactive oxygen species (ROS), decreased coupling of mitochondrial respiration with decreased electron transport capacity, and reduced production of ATP. Transmission electron microscopy (TEM) examination revealed that mitochondrial cristae formation was defective in MA-induced AKI. Gene expression analysis revealed significantly lower ATPase6 levels and protein analysis confirmed that renal mitochondrial proteins of ATP subunits 5A1 and 5C1 were reduced considerably. The increase of NF-kB of the cardiac endothelial cell was observed with immunohistochemistry staining. Our study demonstrated that tubular mitochondrial dysfunction resulting from altered expression of ATP synthase is associated with AKI-induced type 3 cardiorenal syndrome, providing a potential novel target for developing new strategies for preventing and treating CRS3.
Abstract P3186: Mcub Is An Inducible Regulator Of Calcium-dependent Mitochondrial Metabolism
Authors: Huo Jiuzhou , Prasad Vikram , Grimes Kelly , Vanhoutte Davy , Blair Scott , Bround Michael , Bers Donald M , Molkentin Jeff D
Published: October 2023

Summary:
Mitochondria use the electron transport chain to generate high energy phosphate from oxidative phosphorylation of fuel substrates, and the regulation of this process is critical for metabolic homeostasis. Notably, acute influx of Ca
2+
across the inner mitochondrial membrane through the mitochondrial Ca
2+
uniporter (MCU) augments the activity of several electron transport chain dehydrogenases and the F
1
F
0
ATP synthase. However, recent results with deletion of the
Mcu
gene in mice, which prevents acute Ca
2+
influx, shows the unanticipated effect of augmenting total mitochondrial oxidative phosphorylation and weight loss in mice. Here we determined that the MCU complex gene product
Mcub
, which is a direct inhibitor of MCU mediated Ca
2+
influx, is induced by caloric restriction in heart, skeletal muscle, liver and kidney, where it functions as a metabolic activator of mitochondrial fatty acid utilization. Here we performed skeletal muscle-specific deletion of MCUb mice to directly investigate the role of MCUb in substrate utilization. Mice lacking
Mcub
in skeletal muscle showed a metabolic signature of increased pyruvate dehydrogenase activity, increased muscle malonyl CoA, deficient fatty acid utilization, total glucose intolerance, and metabolic syndrome with increased fat deposition. By comparison, fasting induces mitochondrial MCUb expression to reduce mitochondrial Ca
2+
uptake, which results in greater fatty acid oxidation in skeletal muscle. Mechanistically, PDK4 was decreased in muscle of MCUb deleted mice, and overexpression of PDK4 abolished these altered substrate preferences as observed with MCUb deletion in skeletal muscle. Thus, MCUb serves as an inducible control point in regulating skeletal muscle mitochondrial Ca
2+
, which impacts total body metabolism, weight gain and metabolic syndrome.
Abstract P3193: Cardiac Glutamine Metabolism As NAD+ Generating Pathway During Chronic Hypoxia
Authors: Menendez-Montes Ivan , Solmonson Ashley , MAcedo-Pereira Ana Helena , Cardoso Alisson C , Nakada Yuji , Thet Suwannee , DeBerardinis Ralph , Sadek Hesham A
Published: October 2023

Cardiovascular disease morality relies in the inability of the adult mammalian heart to regenerate damaged tissue due to the inability of the adult cardiomyocytes to proliferate. This lack of proliferation in the adult heart is due to an increased DNA damage and activation of the DNA Damage response upon an increase of mitochondrial ROS generation. We have previously shown that modulation of mitochondrial metabolism towards glucose oxidation at expenses of fatty acid utilization can induce adult cardiomyocyte proliferation and support cardiac regeneration. Moreover, by modulating the oxygen levels and, thus, cardiomyocyte metabolism, by chronic hypoxia exposure, we were also able to induce cardiomyocyte proliferation and cardiac regeneration in adult mice. Remarkably, our metabolomic studies of the hypoxic hearts did not show increase of lactate production, a hallmark of hypoxic metabolism in order to regenerate NAD+ required for glycolysis and ATP production under low oxygen conditions. Interestingly, glutamine metabolism is one of the most compensatory pathways in these conditions. Glutamine can be metabolized to its conversion into glutamate, which can enter in the Krebs’ Cycle, but also feed the malate-aspartate shuttle. This shuttle has the ability to generate cytoplasmic NAD+. Here, we hypothesize that, during chronic hypoxia exposure, the heart uses glutamine as a fuel to regenerate cytoplasmic NAD+ as an alternative to lactate production. By means of carbon labeling and metabolic flux studies in vivo, we have proven that glutamine contribution to the malate-aspartate shuttle is enhanced during hypoxia in the heart. Moreover, we have determined that glutamine supplementation during hypoxia in cell culture increases cytoplasmid NAD+ levels, confirming the contribution of glutamine catabolism to NAD/NADH balance during hypoxia. Overall, we show that glutamine metabolism is an important adaptation to hypoxic metabolism in the heart and describe a new mechanism of metabolic rewiring under hypoxia beyond the classic lactate production upon glycolysis upregulation.
Abstract P3196: Voluntary Exercise Training Does Not Prevent High Phosphate Diet-induced Mitochondrial Dysfunction
Authors: Sanders Hannah F , Branch Daniel A , Pepperney Luke J , Oh Jooho , Zonfa Camille E , Liu Rose , Baig Rafia A , Sheridan Molly K , Baskin Kedryn K
Published: October 2023

Inorganic phosphate (Pi) is used extensively as a preservative and a flavor enhancer in processed foods consumed as part of a Western diet. Mice fed high-Pi diet (HP) for 12 weeks display decreased maximal exercise capacity but no change in cardiac function. Expression of fatty acid metabolism genes are decreased and expression of glucose metabolism genes are increased in skeletal muscle after HP diet. Consequently, mitochondrial function is significantly impaired in skeletal muscle from mice fed HP diet. In the current study we test the hypothesis that voluntary exercise training prevents the negative metabolic consequences of HP diet. We housed mice in voluntary wheel running cages and fed them a normal-Pi (NP) or HP diet. HP diet did not alter body weight or composition, and NP- and HP-fed mice voluntarily ran the same distance in wheel cages throughout the study. Using incline treadmill exercise tolerance tests we observed no differences in medium-intensity exercise capacity between NP- and HP-fed mice subjected to voluntary wheel running, contrary to findings in sedentary mice. Despite preserved exercise capacity, muscle mitochondrial function in mice fed HP diet was significantly decreased after 5 weeks, which continued to decline after 12 weeks of HP diet and voluntary exercise. To investigate mechanisms of HP-induced mitochondrial dysfunction we measured expression of key metabolic genes and found that fatty acid metabolism genes were significantly decreased in muscle from mice fed HP diet and subjected to voluntary exercise. These data suggest that voluntary exercise training does not prevent the negative metabolic consequences of consuming a high-phosphate diet. Thus, dietary Pi may represent a novel and modifiable target to reduce physical inactivity associated with the Western diet.
Abstract P3204: Right Ventricle Dysfunction Correlates With Reduced Mitochondrial Respiratory Supercomplex Assembly In Human Hearts With Group II Pulmonary Hypertension.
Authors: Ma Wenzhuo , Zhang Peng , Campbell Kenneth , Choudhary Gaurav , Clements Richard T
Published: October 2023

Introduction:
RV dysfunction and failure is a common consequence of pressure overload associated with all types of pulmonary hypertension. Metabolic and mitochondrial structure and function alterations are further associated with RV dysfunction and failure. Mitochondrial respiratory supercomplexes (mSC) are physically associated assemblies of electron transport chain (ETC) complexes I, III, and IV which are thought to promote improved bioenergetic capacity and efficiency. We previously found reductions in mSC in animal models of PH. It is currently unknown if mSC are reduced in human right ventricle with PH and RV dysfunction.

Methods:
Failing hearts were harvested from patients at the time of transplant (n=20). Sample from the RV were frozen and later mitochondrial isolated and solubilized for Clear-native PAGE (CN-PAGE) to determine complex IV activity. Gels were normalized to Coomassie stained protein via far-red imaging. mSC activity was determined as amount of active CIV in the SC region (>1mD). Only patients with a recent right heart catheterization prior to transplant were used for correlation analysis. Pulmonary artery pulsatility index (PA systolic pressure-PA diastolic pressure/ right atrial pressure (PAPi)) and right atrial pressure to pulmonary capillary wedge pressure (RAP:PCWP) were used as indicators of pulmonary right ventricular dysfunction.

Results:
The majority of patients displayed high mPAP, however PH with RV dysfunction is associated with high mPAP and RAP:PSWP and low PAPi while high mPAP, low RAP:PSWP, and high PAPi indicate PH with normal or preserved RV dysfunction. SC activity as measured as CIV activity in SC region positively correlated with PAPI, (r=.6, P=.004) and negatively with RAP/PCWP (r=
-
.57, P=.009). There was no correlation with non-SC CIV activity as measured by CN-PAGE.

Conclusion:
The severity of RV dysfunction in humans with Group II PH is correlated with reduction in activity of mSC in RV mitochondria
Abstract P3206: Sex-specific Differences In Proteasome Activity And Mitochondrial Function In Mouse Hearts Lacking Natriuretic Peptide Receptor A
Authors: Tiwari Shuchita , Mishra Abhishek , Pasumarthi Kishore
Published: October 2023

Objective:
Atrial natriuretic peptide (ANP) is a cardiac hormone that plays an important role in cardiovascular function and energy metabolism by signaling through natriuretic peptide receptor A (NPRA). Genetic ablation of NPRA in mice resulted in hypertension, cardiac hypertrophy, and fibrosis. The cardiovascular phenotypes were reported to be more severe in male NPRA-knockout (KO) mice. It is known that reductions in proteasomal activities can lead to mitochondrial dysfunction. Similarly, an imbalance between mitochondrial fission and fusion events can impair mitochondrial function. We hypothesized that alterations in proteasomal function and mitochondrial dynamics might form the basis for phenotype differences reported for NPRA-KO mice.

Method/Results:
Non-invasive echocardiography, RT-QPCR, Western blotting, and proteasomal assays were performed to evaluate cardiac functions. Preliminary results revealed significant decreases in the activities of two proteasome subunits (β1 and β5) in male and female NPRA-KO hearts compared to sex-matched wild-type (WT) hearts. Notably, the percent-activity reductions were much higher in male NPRA-KO hearts. Western blotting analysis revealed that the expression levels of mitochondrial fusion proteins (Mfn1, Mfn2, and Opa1) were significantly increased in both male and female NPRA-KO hearts compared to WT controls. However, the expression of fission proteins (Drp1 & MFF) was significantly increased only in male NPRA-KO hearts. Consistent with a more imbalance in mitochondrial dynamics in male NPRA-KO hearts, the expression levels of enzymes that promote fatty acid oxidation (ECHS1 and phospho-PDH) were unfavorably altered in male but not in female NPRA-KO hearts compared to controls. In addition, various echocardiographic and electrocardiographic parameters related to the cardiac function such as cardiac output, diastolic volume, left ventricular wall thickness, QRS duration, and stroke volume, were altered significantly more in male than in female NPRA-KO hearts.

Conclusion:
Our findings suggest that more pronounced sex-specific alterations in proteasome/mitochondrial functions in male NPRA-KO hearts can lead to changes in cardiac metabolism and a severe disease phenotype.
Abstract P3207: Mitoregulin Microprotein Influences Mitochondrial Membrane Integrity And Myocardial Ischemia-Reperfusion Injury
Authors: Stein Colleen S , Zhang Xiaoming , Witmer Nathan , Pennington Edward R , Shaikh Saame R , Boudreau Ryan L
Published: October 2023

Microproteins have emerged as a class of proteins often found to be encoded by misannotated long noncoding RNAs (lncRNAs). We and others discovered that a ubiquitously-expressed “lncRNA” (
LINC00116
) encodes for a highly-conserved mitochondrial transmembrane microprotein that regulates beta-oxidation; we named this protein Mitoregulin (Mtln). In cells and mice, we found that Mtln increases mitochondrial respiratory efficiencies and Ca
2+
retention capacities, while reducing ROS. To date, the molecular function of Mtln remains unclear, with instances of conflicting data across papers. Recently, we found that mitochondria from Mtln-knockout (KO) mice are susceptible to membrane freeze-damage, leading to matrix protein leak during mitochondrial isolation. This appears not to be a distal downstream consequence of chronic Mtln loss, as we have confirmed these findings in cells after acute Mtln knockdown and in a mouse model of inducible cardiac-specific KO of Mtln (AAV-based CRISPR in aMHC-Cas9 mice; ~90% reduction in Mtln). Furthermore, acute re-expression of Mtln using AAV in Mtln-KO mice rescues the membrane deficit. Transmission electron microscopy revealed that acute Mtln knockdown in cells disrupts mitochondrial cristae integrity / density. Given that we previously found that Mtln strongly binds cardiolipin, we assessed the effects of synthetic Mtln peptide on membrane dynamics in mitochondrial-simulated lipid monolayers and found that Mtln modulates lipid packing and mixing and increases monolayer elasticity. Whether these potential direct effects mediate Mltn’s broad positive impact on mitochondria remain unknown. To further explore a role for Mtln in cardiac disease, we subjected Mtln-KO and AAV-Mtln overexpression mice to myocardial ischemia-reperfusion (IR), given Mtln’s influence on mitochondrial Ca
2+
buffering and ROS. Mtln-KO mice showed significantly worse outcomes (increased infarct size, cardiac dilation, and decreased ejection fraction) at 72 h post-IR, whereas AAV-Mtln mice were protected (decreased infarct size and increased ejection fraction), each compared to control littermates. Overall, this work points to new molecular functions for Mtln and highlights its protective (therapeutic) role in IR injury.
Visual, Spatial and Temporal Aporias in the Post-Palestinian Films of Basma Alsharif
Authors: Hole Kristin Lené
Published: October 2023

This article focuses on the films of diasporic Palestinian artist Basma Alsharif, analyzing the visual strategies she mobilizes to negotiate three aporias in Palestinian experience around visuality, space and mobility and temporality. Focusing on Ouroboros (2017) and Home Movies Gaza (2013), I explore the ethical ways she represents a situation of suspended violence without spectacularizing suffering or relying on overdetermined visual conventions. The films evoke both feelings of spatial constraint, and conversely, a diasporic expansiveness that characterizes a Palestinian structure of feeling. Finally, I look at Alsharif’s engagement with the concept of the eternal return, paying particular attention to the ways that it suggests a notion of time not predicated on the unitary, revolutionary (i.e., heteromasculine) subject and proposes a way forward not based on the exclusionary rhetoric of the nation-state. Throughout the article, I put her work in conversation with other female Palestinian filmmakers handling similar themes to identify emerging patterns in how Palestine is portrayed and conceived.
One new species, two new combinations and taxonomic notes on the All-spice genus Pimenta (Myrtaceae) from Hispaniola
Authors: VASCONCELOS THAIS N.C. , LUCAS EVE J. , PEGUERO BRIGIDO
Published: October 2023

One new species and two new combinations are here published as taxonomic updates on the all-spice genus Pimenta (Myrtaceae) for the flora of Hispaniola, Greater Antilles. Pimenta berciliae is a small tree, the type of which was found in the vicinity of the National Botanical Gardens in Santo Domingo. Natural populations of this species are restricted to a small area in Samaná and Cordillera Septentrional, and the preliminary assessment of its conservation status indicates an endangered species. Additionally, Eugenia yumana and Eugenia samanensis are here formally transferred to Pimenta after molecular and morphological analyses demonstrate that they belong to this latter genus. Two new combinations, Pimenta yumana and Pimenta samanensis are here provided. These three additions to the flora of Pimenta in Hispaniola increase the known diversity of the genus on the island and are important to better understand the diversity of the all-spice genus in the region.
Ras-Related Nuclear Protein-Binding Protein 9 Regulates Gastric Cancer Cell Cycle and Apoptosis Through the PTEN/PI3K/AKT Signaling Pathway
Authors: Huang Meihua , Chen Qiansheng , Fan Yong , Liao Quanhuii , Xie Bingkai , Chen Shunqing , Li Shurong , Liang Wei , Zheng Xiaoling
Published: October 2023

We investigated the impact of RanBP9 on cell cycle progression and apoptosis in gastric cancer cells. RanBP9 expression was analyzed in 38 clinical gastric cancer tissues using Western blotting. Lentiviral transfection was utilized to establish GES-1 gastric cancer cell models that
either overexpressed or silenced RanBP9. Cell proliferation and apoptosis were assessed using MTT and TUNEL staining assays, respectively. Apoptosis-related factors were analyzed by Western blotting and qRT-PCR. Flow cytometry and qRT-PCR were employed to evaluate cell cycle progression and
the mRNA levels of CDK4/CyclinD1. The PTEN/PI3K/AKT signaling pathway was examined by Western blotting. We observed a significant reduction in RanBP9 expression in gastric cancer tissues. Overexpression of RanBP9 in GES-1 cells suppressed cell activity, enhanced apoptosis, increased Caspase3
expression and the Bax/Bcl-2 ratio, and decreased CDK4 and CyclinD1 expression, thereby preventing S phase entry. Conversely, knockdown of RanBP9 yielded opposite results. Furthermore, we found that RanBP9 negatively regulated the PTEN/PI3K/AKT pathway. Our findings demonstrate low expression
of RanBP9 in gastric cancer tissues and cell lines. We have also established that RanBP9 negatively regulates the PTEN/PI3K/AKT pathway, resulting in cell cycle prolongation and promotion of apoptosis in GES-1 cells.
Multifunctional Biocompatible Rhinacanthus nasutus Based TiO2-Doped CeO Nanoparticles Synthesis and their Biomedical Evaluations; In-Vitro/In-Vivo Approach
Authors: Rajaduraipandian S. , Amutha E. , Sabeena G. , Ponnani Kaja Mideen M. , Mercy A. , Gandhimathi S. , Annadurai G. , Mariselvam R. , Alahmadi Tahani Awad , Alharbi Sulaiman Ali
Published: October 2023

The work intended to evaluate the potential wound healing properties of TiO2 doped CeO nanoparticles with the assistance of Rhinacanthus nasutus. Furthermore, an assessment was conducted on the nanoparticles to determine their antioxidant, cytotoxic, anti-diabetic,
and anti-inflammatory inhibitory properties, as well as their toxicity in albino rats. The nanoparticles were synthesized in the green method and subjected to characterization through various methods including UV-visible spectroscopy, SEM for morphological study, FTIR to identify functional
groups, XRD, and for elemental analysis EDAX. The morphology of the observed nanoparticles were predominantly spherical, exhibiting an agglomerated structure. The findings indicated that approximately 49% of the nanoparticles exhibited DPPH antioxidant activity, as determined by an IC50 value
of 2.8 g/mL. The nanoparticles exhibited cytotoxicity in the brine shrimp lethality assay when administered at a concentration of 50 g/mL. Additionally, they displayed notable inhibitory activity against-amylase, with an IC50 value of 2.981 g/mL. The Ames test yielded negative results, suggesting
that the nanoparticles exhibited non-toxic properties. In general, the study substantiated the prospective biological uses of TiO2-doped CeO nanoparticles.