bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2067: Depleted Cardiac Energy Reservoirs And Type 2 Diabetes: The Unfulfilled Role Of Endogenous Creatine
Authors: White Melanie Y
Published: November 2022

To maintain contractility, the heart turns over the cellular ATP pool six times
per
minute, with up to half replenished
via
the creatine:phosphocreatine system (Cr:PCr). This ‘safe’ energy reservoir arises from endogenous sources, through biosynthesis in the kidney and liver for delivery to the heart. Exogenous PCr drastically improves contractility following myocardial ischemia/reperfusion injury (I/R), as exemplified by cardioplegic PCr supplements provided during coronary artery bypass surgery. Considering clinical outcomes following I/R are typically worse in patients with Type 2 Diabetes (T2D), we hypothesized that T2D comorbidities that impact physiology of the kidney and liver may impair endogenous production of Cr and limit cardiac Cr:PCr. We employed a rat model that combined obesity and steatosis (high-fat diet), with pancreatic insufficiency (streptozotocin), to produce elevated blood glucose, serum triglycerides and reduced insulin sensitivity, consistent with a T2DM phenotype. Heart, liver, kidney and blood were sampled from T2D rats for histology and metabolomics by targeted mass spectrometry. Renal microanatomy showed little variation in response to T2D, while nuclear displacement and vacuolation was apparent in liver sections. We identified no change in renal Cr substrate guanidinoacetate (GAA) (0.7996±0.09;
n
-fold±SEM,
p
-value=0.524) or the serum GAA levels (0.95±0.13;
n
-fold±SEM,
p
-value=0.993). In the liver, GAA levels were depleted (0.468±0.04;
p
<0.0001), with a comparable reduction of hepatic creatine abundance (0.391±0.07;
p
=0.003). Cardiac levels of both Cr and PCr were significantly depleted in the heart (Cr: 0.392±0.02,
p
<0.0001; and PCr: 0.642±0.07,
p
=0.02). To confirm that this decrease in cardiac Cr:PCr did not simply reflect increased metabolism, we monitored cardiac creatinine and found only a modest decrease in T2D hearts (0.783±0.05;
p
=0.003), confirming a depletion in Cr bioavailability. In summary, hepatic derangement indicative of steatosis promoted impaired creatine biosynthesis to limit availability of Cr:PCr for restoration of ATP in the diabetic heart. In the absence of adequate ‘safe’ energy reservoirs, such hearts are compromised further following I/R injury.
Abstract P2073: Rbx2-CRL5 Mediates Mitochondrial Ubiquitination And Mitophagy To Maintain Myocardial Homeostasis Independent Of Parkin
Authors: Wang Wenjuan , Li Ermin , Zou Jianqiu , Qu Chen , Ayala Juan , Pratt Kayla , Liu Jinbao , Li Jie , Su Huabo
Published: November 2022

The ubiquitin (Ub) ligase Parkin has been recognized as a central player in mitophagy. In contrast to its presumed importance in the heart, ablation of Parkin fails to impact homeostatic mitophagy and cardiac function in both young and aging mice, suggesting compensation from other mitochondrial Ub ligases. However, the identities of such additional mitochondrial Ub ligases, as well as their physiological significance in the heart, remain unclear. Here, we demonstrate that Cullin-RING Ub ligase 5 (CRL5) plays a key role in the control of mitochondrial homeostasis in cardiomyocytes. CRL5 belongs to the family of Cullin-RING Ub ligases (CRLs) and consists of the RING box protein RBX2, scaffold protein Cullin 5 (Cul5), and one of 38 substrate receptor proteins. Analysis of the mitochondrial outer membrane-interacting proteome revealed a robust association of CRLs with mitochondria. Pharmacological and genetic perturbations of CRLs inhibited mitochondrial ubiquitination and mitophagy. Subcellular fractionation, live-cell imaging and immunogold electron microscopy further revealed that RBX2 and Cul5, two core components of CRL5, are quickly recruited to mitochondria upon mitochondrial depolarization. Depletion of RBX2 or Cul5 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial function, and increased cell death in cardiomyocytes. Importantly, RBX2 deficiency did not alter Parkin expression, had a more robust inhibitory effect on mitochondrial ubiquitination compared to Parkin deficiency, and remained effective in the absence of Parkin. Moreover, loss of RBX2 attenuated Parkin-mediated ubiquitination, suggesting its dominant, Parkin-independent actions in mitochondria.
In vivo
, deletion of RBX2 in adult mouse hearts suppressed mitophagic activity and provoked accumulation of damaged mitochondria in myocardium, leading to rapid development of dilated cardiomyopathy and heart failure. Finally, RBX2 was upregulated in pressure overloaded mouse hearts but downregulated in human failing hearts, further supporting its protective role in the heart. Taken together, our data allow us to propose RBX2-CRL5 as a mitochondrial Ub ligase and a physiological regulator of mitochondrial and cardiac integrity.
Abstract P2076: β-hydroxybutyrate Administered At Reperfusion Alleviates Myocardial Ischemia-reperfusion Injury By Enhancing Autophagy And Mitochondrial Homeostasis
Authors: Chu Yuxin , He Lihao , Hua Yutao , He Jin , Chen Yunxi , Benavides Gloria , Darley-Usmar Victor , Young Martin E , Zhang Cheng , Xie Min
Published: November 2022

Introduction:
ST-elevation myocardial infarction (STEMI) is the leading cause of heart failure. Ischemia/reperfusion (I/R) injury after revascularization contributes ~50% of infarct size, which we don’t have a clinical therapy. β-hydroxybutyrate (β-OHB) is a metabolite that increases during stress. In addition to providing energy, it serves as a signaling molecule (such as histone deacetylase inhibitor) to regulate many protective processes. Increasing the β-OHB level before I/R is cardiac protective. However, it has not been tested when administered at reperfusion.

Hypothesis:
The physiological concentration of β-OHB administered at the time of reperfusion reduces I/R injury by enhancing autophagy and mitochondrial homeostasis.

Methods:
C57BL6 wild-type mice were randomized into 2 groups and subjected to I/R surgery (I: 45 mins; R: 24 hrs). At reperfusion, normal saline or 10 mmol/kg of β-OHB (4-6 mM blood level) was given intraperitoneally. Echo was used to evaluate cardiac functions. We measured Infarct size, autophagic flux, and mitochondrial DNA levels in the heart. CAG-RFP-GFP-LC3 (RFLC3) mice were used to detect autophagic flux. Neonatal rat ventricular myocytes (NRVMs) were subjected to simulated I/R to test the effect of β-OHB
in vitro
. Small interfering RNA of ATG7 gene was used to assess the role of autophagy. Seahorse assay was performed to evaluate mitochondrial function after I/R.

Results:
β-OHB gave at reperfusion reduced infarct size by 50% (N=15 in control and N=11 in β-OHB group), and preserved systolic function in mice. In the border zone, the LC3II and the mtDNA level increased, and β-OHB increased autophagic flux in RFLC3 mice (N=5). In NRVMs subjected to I/R, β-OHB increased histone acetylation, cell viability, and maintained mitochondrial homeostasis (mitochondrial mass, membrane potential), and reduced ROS generation (N=4-6). Seahorse assay showed β-OHB enhanced ATP production after I/R. ATG7 knockdown blocked the protective effect of β-OHB in NRVMs.

Conclusions:
β-OHB administered at reperfusion reduces infarct size by increasing autophagic flux and maintaining mitochondrial homeostasis. Since β-OHB has been used in heart failure patients safely, it is a viable therapeutics to reduce infarct size in STEMI patients.
Abstract P2080: Role Of Atg9 Proteins In Regulating Autophagosome Formation
Authors: Diao Rachel Y , Liang Wenjing , Chi Liguo , Fang Xi , Gustafsson Asa
Published: November 2022

Cardiac myocytes require large amounts of energy generated by mitochondria to sustain contraction, and there is a strong link between mitochondrial dysfunction and heart disease. Defective mitochondria are less efficient at generating ATP, produce excessive reactive oxygen species, and release pro-apoptotic proteins, all of which can lead to loss of cardiac myocytes and reduced ability to sustain contractile function. Therefore, mitochondrial quality control is critical in preventing mitochondria from causing harm to the cell. Selective autophagy of the mitochondria (mitophagy) is the primary mechanism responsible for eliminating dysfunctional mitochondria. Despite intense research focus on mitophagy over the past decade, little is still known about early events in the pathway, and the signals that orchestrate the selective recruitment of the autophagic machinery to dysfunctional mitochondria are unclear. It is known that autophagosome formation requires Atg9, which is embedded in small Golgi-derived vesicles. To date, most data on Atg9 are from yeast and our knowledge of mammalian Atg9a and Atg9b functions in cells and tissues are still limited. Here, we found that Atg9a and Atg9b are expressed in the heart and that both proteins are increased in the infarct border zone after ligation of the LAD. We also discovered that Atg9a and Atg9b are present on distinct vesicles in myocytes. Atg9b-positive vesicles are localized throughout the cytosol, whereas Atg9a-positive vesicles are associated with the mitochondria even under basal conditions. This suggests that they might have distinct functions and that Atg9a may play a more selective role at the mitochondria. Interestingly, Atg9a, but not Atg9b, is degraded by the proteasome upon activation of mitophagy, a potential indication that this may be a mechanism to prevent excessive mitophagy. Overall, our findings suggest that Atg9a and Atg9b play distinct roles in autophagy and mitophagy and that the mitochondrial localization of Atg9a allows it to initiate autophagosome formation more efficiently during mitophagy.
Abstract P2084: Determining The Role Of Rap1b In Megakaryocyte Survival
Authors: Mattmiller Audrey A , Wisinski Jaclyn A
Published: November 2022

Platelet counts are tightly regulated through production by megakaryocytes and clearance by hepatocytes. Thrombotic risk and poor cancer prognosis correlate with platelet counts on the high and low ends of the normal range, respectively. Therefore, understanding the factors that regulate megakaryocyte survival and function are important for human health. The small GTPase, Rap1b increases in abundance as megakaryocytes mature and mediates survival in other cell types by preventing apoptosis. We hypothesize that disruption of the Rap1b gene in the megakaryocytic leukemia cell line, DAMI, will limit cell survival by increasing susceptibility to apoptosis. We used CRISPR/Cas to specifically disrupt the Rap1b gene, resulting in no detectable Rap1b protein in DAMI cells. Compared to wild-type DAMI cells, Rap1b knockout cells had nearly a 50% reduction in survival as measured by metabolic activity using an MTT-assay. To assess apoptosis susceptibility, we monitored mitochondrial membrane potential using the fluorescent sensor JC-1 and caspases activity using the Fluorochrome Inhibitor of Caspases (FLICA). These results suggest that Rap1b promotes megakaryocyte survival.
Abstract P2086: Wirelessly Powered Miniaturized Intravascular Pacemaker Based On Inductive Coupling
Authors: Cui Qingyu , Abiri Parinaz , Cavallero Susana , Tai Yu-Chong , Hsiai Tzung K
Published: November 2022

Introduction:
Despite unparalleled advantages, clinical lead-associated complications of the conventional pacemakers remain to compromise patient safety and survival, accounting for nearly 10% of all implants. The leadless pacemakers from Medtronic or St. Jude Medical are revolutionary products, however, the integrated battery introduced new clinical constraints and health risks. Therefore, developing leadless pacemakers incorporating wireless powering owns special clinical significance to further the miniaturized integration and reduce the repetitive mechanical workload for the hearts.

Methods and Results:
Hereby, we achieved the leadless intravascular miniaturized pacemaker powered by electromagnetic inductive coupling. The miniaturized assembling was built up from flexible circuit boards, integrating with receiver coils and tiny electrical components for the RF-DC converting. The substrate circuit board will be bent into a cylinder with a diameter of 2.7 mm and filled with PDMS to achieve the electrical isolation and form a narrower tip for easy insertions (Figure 1a,b). The voltage of DC pulse converted at the wireless pacemaker depends on the DC voltage for the RF amplifier and the gap between the coils, ranging from 0.5V to 5V or higher (Figure 1c). After inserting the pacemaker into the anterior vein, the pacing demonstration showed the stimulation spikes and the following regular cardiac Q-R-S-T waves, indicating the pacemaker can stimulate the intrinsic cardiac rhythms (Figure 1d,e).

Conclusions:
The proposed medical device reveals the potentials of cardiac pacing from the anterior veins and paved the way to further device miniaturization for the intravascular deployment.
Abstract P2104: Adeno-associated Virus Serotype 5 Is A Suitable Vector For S100a1-based Gene Therapy Of Post-ischemic Chronic Cardiac Dysfunction
Authors: Kehr Dorothea , Salatzki Janek , Krautz Birgit , Gao Erhe , Varadi Karl , Birkenstock Jennifer , Schlegel Philipp , Müller Oliver , Raake Philip W , Egger Michael , Koch Walter J , Riffel Johannes , André Florian , Katus Hugo A , Frey Norbert , Jungmann Andreas , Busch Martin , Pfannkuche Helga , Most Patrick
Published: November 2022

Introduction:
For S100A1-based heart failure gene therapies, AAV9 and 6 have shown efficacy in pre-clinical large animal studies. As AAV9 has shown concerning signs of toxicity in clinical studies and AAV6 displays poor production yields, there is need for a novel safe and cardiac-specific AAV serotype.

Hypothesis:
We hypothesized that in a pig model the safety proven and scalably manufacturable AAV5 may be a suitable vector for S100A1-based gene therapy of post-ischemic cardiac dysfunction.

Methods:
AAV production, 2h balloon-occlusion of the LCX, retrograde cardiac gene delivery, cardiac MRI, late gadolinium enhancement (LGE), global T1 relaxation, qPCR, RNA-Seq, WGCNA, KEGG, Reactome, LAD-ligation mouse model

Results:
In a comparative study of AAV5-, 6- and 9-luciferase (luc) in healthy farm pigs (n=5 each; 1x10
13
vgc/pig), AAV5 achieved a more homogeneous cardiac apical-basal transduction pattern than AAV6 with a higher luc activity than AAV9. In a clinically relevant endpoint driven study, we demonstrated a significant improvement in EF (+19 ± 5 %) 12 weeks after retrograde AAV5-
S100A1
gene delivery compared to AAV5-luc in infarcted pigs (n=4 each; 1x10
13
vgc/pig). Moreover,
S100A1
-treated pigs showed significantly less infarct extension (-0.5 ± 0.3 g vs. 5 ± 1.3 g (luc)) measured by cardiac MRI. There were no unfavorable alterations in blood chemistry or ECG.
S100A1
expression was predominantly contained to the heart. The WGCNA unveiled a significant correlation between the improved EF and a suppression of inflammatory and immunological pathways (r=-0.96, p < 0.01) and between the absent infarct extension and enhanced activity of cardioprotective signaling (r=-0.82, p < 0.05). With injections of 2х10
11
vgc of AAV5-
S100A1
or AAV5-gfp (n=4 each) into the remote myocardium in the mouse model, we confirmed a significant improvement in FS (+43.8 ± 8.8 %, vs. gfp) and suppression of inflammatory gene expression including i.e., IL1b or TNFa by S100A1.

Conclusion:
We conclude that AAV5 is suitable for S100A1-based gene therapy of post-ischemic cardiac dysfunction and that this vector/target combination can help accelerating the way towards a clinical trial. We also found novel signaling pathways that may be involved in S100A1’s therapeutic actions.
Abstract P2106: Arrhythmia As Cause For The Development Of The Takotsubo Syndrome In A Patient-specific Atrial Stem Cell Model
Authors: Brandt Charlotte , Huebscher Daniela , Syed-Ali Gideon , Nikolaev Viacheslav O , Streckfuss-Boemeke Katrin
Published: November 2022

Background and Aims:
The Takotsubo syndrome (TTS) is characterised by an acute left ventricular dysfunction without exhibiting signs of stenosis. TTS affects mainly the left ventricle, however, its pathophysiology comprises transient impairments in left atrium functions with a prevalence of atrial fibrillation (AF) of around 18% correlating with higher ventricular heart rhythm disorders. In this study, we aimed to prove the hypothesis that molecular and cellular arrhythmic events contribute to the development of TTS under catecholamine stress and to test if the currently clinically used β-blockers (Metoprolol) are suitable for Takotsubo cardiomyopathy in vitro.

Methods and Results:
We generated induced pluripotent stem cell-derived atrial cardiomyocytes (TTS-iPSC-
a
CMs) from TTS patients, confirmed atrial marker expression (MLC2a, PItX2, NR2F2), and treated the cells with catecholamines to mimic TTS-phenotype. Using a cytosolic Förster resonance energy transfer (FRET) based cAMP sensor, we tested the activity of phosphodiesterases (PDEs) in TTS-iPSC-aCMs, which are besides β-ARs main players involved in cAMP signaling in CMs. We observed that after β-AR stimulation, the strong effects of the PDE4 family in the cytosol of atrial controls were significantly decreased in aCMs of the TTS patients. This effect was completely rescued after application of PDE4 activator MR-L2 and is in line with the previously described downregulation of PDE4 in human atrial myocardium of AF patients. In functional studies, isoprenaline-induced increase in systolic Ca
2+
transient amplitude was more pronounced in TTS iPSC-aCM compared to controls. These effects were rescued by both, the clinically approved β-blocker Metoprolol as well as the PDE4 activator MR-L2. To analyse arrhythmic events in atrial TTS CMs, we performed confocal Ca
2+
measurements and demonstrated that the diastolic sarcoplasmic reticulum Ca
2+
leak was increased in iPSC aCMs of TTS patients compared to control under basal conditions and after Iso-treatment. In addition, TTS patients displayed faster Ca
2+
kinetics compared to control cells, already under basal conditions. These results were underlined on a molecular level by increased phospholamban phosphorylation in TTS iPSC-aCM .Subsequent treatment with the beta-blocker Metoprolol rescued the Ca
2+
kinetic parameters in all cell lines.

Conclusion:
In conclusion, we were able to draw a comprehensive picture on the role of the arrhythmia in the development of TTS. We found TTS-specific differences with reduced PDE4 activity, elevated arrhythmic events and enhanced reactions to catecholamines, which could be rescued by the clinically approved drug Metoprolol and partly by the PDE4 activator MR-L2. Therefore, Metoprolol as well as MR-L2 demonstrate promising effects as patient-specific therapeutic target for TTS.
Abstract P2110: Myosin-7 E848G Mutation Reduces Contractility And Activates P53-associated Intrinsic Apoptosis In Patient-derived Induced Pluripotent Stem Cell Model Of Hypertrophic Cardiomyopathy
Authors: Loiben Alexander , Friedman Clayton , Chen Wei-Ming , Chao Leslie , Yang Kai Chun
Published: November 2022

Introduction:
Familial hypertrophic cardiomyopathy (HCM), affecting 1 in 500 adults, is characterized by idiopathic thickening of the heart and impaired systolic function. Mechanisms through which cardiac sarcomeric mutations manifest in HCM are poorly understood.

Hypothesis:
We previously identified a novel MYH7 E848G mutation associated with HCM that manifested as left ventricular hypertrophy, diffuse fibrosis, and severe systolic dysfunction. We hypothesize that the E848G variant impairs tissue contractility and causes cell death in a dose-dependent manner.

Methods:
We created MYH7-EGFP expressing cardiomyocytes (CMs) with
MYH7
wt/wt-EGFP
,
MYH7
wt/E848G-EGFP
, or
MYH7
E848G/E848G-EGFP
alleles using patient-derived induced pluripotent stem cells (hiPSCs), CRISPR-Cas9 gene-editing, and standard differentiation protocols. CMs were cocultured with stromal cells to create 3D engineered heart tissues (EHTs) or cultured as a monolayer in high calcium stress media. EHTs were enzymatically digested and GFP+ cardios were sorted for analysis.

Results:
Monolayer
MYH7
wt/E848G-EGFP
CMs had higher TUNEL
+
percentage (13.1 ± 2.1%) at d33 and lower cell survival (52.0 ± 6.6% of d30 seeded) at d42 relative to
MYH7
wt/wt-EGFP
(6.6 ± 2.6%; 107 ± 2.1%). Screening revealed elevated levels of proteins associated with intrinsic apoptosis (p53, 1.93 ± .03-fold; p21, 1.66 ± .10-fold; Bax, 1.56 ± .11-fold) in
MYH7
wt/E848G-EGFP
CMs at d35 relative to
MYH7
wt/wt-EGFP
.
MYH7
wt/E848G-EGFP
(n = 20; 67.5 ± 9.0 μN) had reduced maximum active twitch force relative to
MYH7
wt/wt-EGFP
(n = 19; 226 ± 17 μN) 14 days post-cast.
MYH7
wt/E848G-EGFP
EHTs had lower percentage of EGFP
+
cells (13.5 ± 0.8%) 7 days post-cast relative to
MYH7
wt/wt-EGFP
(25.8 ± 0.8%). EGFP
+
cells sorted from 7 day post-cast
MYH7
wt/E848G-EGFP
EHTs had elevated
TP53
transcription (1.67 ± .29-fold) relative to
MYH7
wt/wt-EGFP
.

Conclusion:
These results suggest
MYH7
E848G mutation reduces sarcomere contractility and induces p53-associated cell death. High calcium stress media induces cell death that likely contributes to contractile dysfunction seen in MYH7 E848G HCM. Ongoing studies will elucidate mechanisms through which E848G activates intrinsic apoptosis.
Abstract P2111: Hutchinson Gilford Progeria Patient-specific Induced Cardiomyocyte Model Carrying Lamin A Gene Variant C.1824 C > T
Authors: Perales Selene G , Rajasingh Sheeja , Sigamani Vinoth , Gurusamy Narasimman , Rajasingh Johnson
Published: November 2022

Background:
Hutchinson-Gilford progeria (HGP) is a rare genetic disorder in which children age rapidly. They suffer from atherosclerosis, cardiovascular diseases, and strokes. The LMNA variant c.1824C>T accounts for ~90% of HGP cases. This variant causes an abnormal Lamin A protein called progerin. The detailed molecular mechanisms of Lamin A in the heart remain elusive due to the lack of appropriate
in vitro
models.

Hypothesis:
We hypothesize that HGP patient’s induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMCs) will provide a model platform to study the cardio-pathologic mechanisms associated with progeria.

Methods and Results:
We performed
in silico
analysis of LMNA variant c.1824C>T to determine its effects on Lamin A structure and stability. For
validation
, skin fibroblasts (SFs) from a de-identified HGP patient (hPGP1, proband) and parents were obtained from the Progeria Foundation. Through Sanger sequencing
(Fig 1A)
and restriction fragment length polymorphism, with enzyme
EciI
, targeting Lamin A, we characterized hPGP1-SFs as heterozygous mutants for variant c.1824 C>T. Then, we reprogrammed the three SFs into iPSCs
(Fig 1B)
using a safe mRNA-based reprogramming method
,
and differentiated them into iCMCs, which gained beating on day 7. We found that hPGP1-iCMCs
(Fig 1C)
had an irregular contractile function by Particle Image Velocimetry Analysis and decreased cardiac-specific gene and protein expressions by qRT-PCR and Western Blot.

Conclusions:
We successfully generated iCMCs from a progeria patient carrying LMNA variant c.1824C>T. These iCMCs were found to be functionally and structurally defective when compared to normal iCMCs. Our
in vitro
model will help elucidate the role of Lamin A and the cardio-pathologic mechanisms associated with progeria.
Abstract P2113: A Novel Patient-derived Stem Cell Model Of Restrictive Cardiomyopathy Exhibits Impaired Cardiomyocyte Relaxation Secondary To Increased Myocardial Calcium Sensitivity
Authors: Staudt David , Serrano Ricardo , Feyen Dries , Mercola Mark
Published: November 2022

Introduction:
Restrictive Cardiomyopathy (RCM) is a rare disease characterized by profound, isolated defects in myocardial relaxation, normal or near-normal myocardial wall thickness, and preserved contractile function. Outcomes for patients with RCM are poor, with increased morbidity and mortality compared to other forms of cardiomyopathy. In some patients, RCM can be caused by genetic mutations, notably in genes encoding for components of the sarcomere. While many mutations have been described in RCM patients, the molecular and cellular mechanisms leading from these mutations to the patient’s physiological phenotype are poorly described.

Hypothesis:
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from RCM patients will recapitulate the myocardial relaxation defects seen in RCM and provide a novel
in vitro
model for investigating the molecular mechanisms underpinning this disease.

Methods:
Induced pluripotent stem cells (iPSCs) were derived from a pediatric patient with RCM caused by a TNNT2 R94C mutation. CRISPR was used to correct the mutation, and cellular mechanics and calcium handling properties were compared to the patient line using a novel high-throughput traction-force imaging platform. Transcriptional profiles were compared using RNA-seq.

Results:
RCM cells carrying the TNNT2 R94C mutation show increased calcium sensitivity leading to increased resting tension at baseline, as well as prolonged calcium kinetics leading to prolonged relaxation times. Gene expression profiling reveals changes in expression of ECM genes, which may further contribute to the impaired myocardial relaxation seen in RCM.

Conclusions:
Patient-derived hiPSC-CMs from an RCM patient recapitulate the relaxation defects seen in RCM and highlight the central role of increased calcium sensitivity in impaired myocardial relaxation.
Abstract P2116: Hyperglycemia Mediated Changes In A Human Cardiomyocyte Cell Model
Authors: Joddar Binata , Singh Irtisha , Chattopadhyay Munmun , Thakur Vikram
Published: November 2022

Introduction:
Hyperglycemia-mediated cardiac dysfunction is a critical initiator in the development of vascular complications, which, in turn, leads to cardiac fibrosis. In this study, we investigated the role of the Hippo signaling pathway in cardiomyocytes to study the complex signaling network of YAP1/TAZ on fibrotic and vascular inflammatory mediators in hyperglycemic condition. This in-vitro study demonstrated that YAP1/TAZ signaling is highly activated in the hyperglycemic cardiomyocytes. To further investigate the differentially expressed genes that are related to inflammation and fibrosis, RNA-sequencing studies were employed.

Methods:
To investigate the effects of hyperglycemia-mediated changes in cardiomyocytes, we used human AC16 cells cultured in-vitro under normoglycemic (5 mM D-Glucose) and hyperglycemic (50 mM D-glucose) conditions. After 24-hours of hyperglycemic insult, cells were collected and processed for RNA-seq studies. Furthermore, we also performed Western Blot analysis to evaluate the protein expression of YAP1/TAZ under hyperglycemia induced stress conditions.

Results:
Our study showed a significant upregulation of the protein expression of the YAP1/TAZ pathway in hyperglycemic cardiomyocytes. RNA-seq studies revealed differentially expressed genes (DEG) in the hyperglycemic condition in comparison with the normoglycemic condition. Among the extracellular matrix proteins, the following ECM and related markers were significantly upregulated including MMP3, TNC, TGF-beta1 and 2, COL4A1, FN1 and FGF-2. Altered expression of inflammatory mediators included the following markers, IL-6, CXCL10, CXCL12, CCL2 and VEGF-C. In addition, the following transcriptional co-activators were also significantly upregulated, including EPHA2 and MYOCD.

Conclusions:
This study suggests that changes in YAP1/TAZ signaling increases vascular inflammation in response to hyperglycemia. This also leads to increased expression of inflammatory mediators as shown by our results. Thus, the inhibition of the YAP1/TAZ pathway may prevent and improve hyperglycemia associated vascular damage and inflammation.
Abstract P2120: Microdystrophin Design Comparisons In Dystrophin-Deficient Cardiomyocytes
Authors: Eguchi Asuka , Gonzalez Adriana Fernanda , Torres-Bigio Sofia , Bhimaraju Sriram , Birnbaum Foster , Blau Helen M
Published: November 2022

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Dilated cardiomyopathy is the leading cause of death in DMD patients. Preclinical data show that smaller versions of dystrophin, called microdystrophins, can ameliorate disease progression. These transgenes, amenable to packaging into adeno-associated virus, have shown to be effective in improving skeletal muscle function; however, the functional benefit of microdystrophins in delaying heart failure is unknown. To identify the design principles that are important for addressing DMD heart dysfunction, three microdystrophin variants currently in clinical trials were tested in cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) with deficiencies in dystrophin. Previously, we reported that mechanical contraction and calcium handling are impaired in DMD iPSC-CMs. To test the functional benefit of the microdystrophins, we performed traction force microscopy, that provides multiparametric measurements of contraction, and calcium imaging with a ratiometric calcium-binding dye. Our results show a partial rescue of contractile deficits and aberrant calcium handling, underscoring the need for new design variants that address cardiomyocyte dysfunction.
Abstract P3003: Endothelial-Specific Expression Of Cidec Improves High-Fat Diet-induced Vascular And Metabolic Dysfunction
Authors: Balakrishnan Bijinu , Gupta Abhishek , Sharma Vishva M , Slayton Mark , Gentner Kailey , Karki Shakun , Muturi Harrison , Najjar Sonia M , Loria Analia S , Gokce Noyan , Puri Vishwajeet
Published: November 2022

Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), originally identified to be a lipid-droplet associated protein in adipocytes, positively associates with insulin sensitivity. Recently, we discovered that it is expressed abundantly in human endothelial cells (ECs) and regulates vascular function in obesity. The present study was designed to characterize the physiological effects and molecular actions of endothelial CIDEC in the control of vascular phenotype and whole-body glucose homeostasis. To achieve this, we generated a humanized mouse model expressing endothelial-specific human CIDEC (E-CIDECtg). E-CIDECtg mice exhibited protection against HFD-impaired glucose intolerance, insulin resistance and dyslipidemia. Moreover, these mice displayed improved insulin signaling and eNOS activation, enhanced endothelium-dependent vascular relaxation and improved vascularization of adipose tissue, skeletal muscle, and heart. Mechanistically, we identified a novel interplay of CIDEC-VEGFA-VEGFR2 that reduced VEGFA and VEGFR2 ubiquitin-mediated degradation thereby increasing VEGFR2 activation. Overall, our results demonstrate a protective role of endothelial CIDEC against obesity-induced metabolic and vascular dysfunction, in part, by modulation of VEGF signaling. These data suggests that CIDEC may be considered as a potential future therapeutic target for mitigating obesity-related cardiometabolic disease.
Abstract P3004: Small Leucine-Rich Proteoglycan Asporin Represses Smooth Muscle Cell Proliferation And Regulates Pulmonary Hypertension Development
Authors: Medzikovic Lejla , Ruffenach Gregoire , HONG JASON , Aryan Laila , Eghbali Mansoureh
Published: November 2022

Pulmonary arterial hypertension (PAH) is a devastating pulmonary vascular lung disease characterized by increased pressure in the pulmonary arteries leading to cardiac right ventricular (RV) hypertrophy, RV failure, and ultimately death. Current PAH therapies merely improve symptoms temporarily and patients often need lung and heart transplantation. As such, there is dire need for new insights into PAH pathophysiology to find novel therapeutic approaches. A key mechanism driving PAH is medial hyperplasia resulting from enhanced pulmonary arterial smooth muscle cell (PASMC) proliferation. Using an unbiased bioinformatics approach in an online-available microarray expression dataset of dissected pulmonary arteries (PA) from PAH patients, we discovered a novel gene, small leucine-rich proteoglycan Asporin (Aspn), which is highly and significantly upregulated in PAH compared to otherwise-healthy donor PA. Further bioinformatic analysis at a single-cell level in an online human lung dataset revealed that Aspn is most highly expressed in pulmonary SMC. We validated higher Aspn mRNA expression in human lungs and PASMC from PAH patients compared to controls, supporting our bioinformatics approach. Moreover, Aspn protein expression is elevated in PASMC in lung tissue sections from PAH patients compared to donors
.
Functionally, silencing Aspn in PASMC from PAH patients promoted proliferation. To find potential downstream mediators of Aspn, STRING database analysis revealed that Aspn binds to the pro-proliferative mediator TGFβ1. Accordingly, TGFβ1 downstream mediator p-SMAD2 is enhanced upon Aspn silencing in PASMC.
In vivo
, intratracheal delivery of Aspn siRNA in rats aggravated Sugen/Hypoxia-induced PH, as assessed by RV systolic pressure, echocardiography, and pulmonary vascular remodeling. Taken together, we hypothesize that elevated Aspn expression in PAH is a compensatory mechanism that inhibits PASMC proliferation, leading to decreased PA vascular remodeling, and that Aspn may be a novel therapeutic target against PAH.
Abstract P3008: Endothelial Stearoyl-CoA Desaturase-1 Catalyzes Fatty Acid Metabolites To Mitigate Arterial Inflammation And Stiffness
Authors: Cavallero Susana , Roustaei Mehrdad , Blazquez-Medela Ana M , Satta Sandro , Phan Henry , Baek Kyung In , Li Rongsong , Hsiai Tzung K
Published: November 2022

Introduction:
Vascular metabolic plasticity is intimately linked with endothelial homeostasis. Stearoyl-CoA desaturase-1 (SCD1), the enzyme that catalyzes the saturated long-chain fatty acids (FA) into monounsaturated FA (MUFA), is responsive to pulsatile shear stress (PSS), and is associated with cancer cell metabolism. We assessed whether exercise-mediated PSS activates PPARγ-SCD1 to mediate anti-inflammatory metabolites for vascular protection.

Methods and Results:
Human aortic endothelial cells (HAEC) were subjected to PSS (τ
avg
= 23±8 dyne/cm
2
at 1 Hz) for 4 hours, followed by metabolomic analysis. SCD1-mediated fatty acid metabolites, azelaic acid (AZA) and oleic acid (OA), were identified to mitigate NFκB luciferase activity and monocyte binding (
p
< 0.05, n= 5, vs. static condition). PSS upregulated PPARγ and SCD1 mRNA expression, whereas knockdown of PPARγ using siRNA downregulated SCD1. Wild type mice (C57BL/6J) undergoing 2-wk voluntary exercise developed increased perinuclear SCD1 expression in the aortic endothelium (
p
< 0.05, n=6, vs. sedentary controls), whereas SCD1 expression was reduced in PPARγ -dominant negative mice following exercise. LDLR
-/-
mice with endothelial-specific SCD1 deletion (SCD1
EC-KO
: Cdh5
Cre
, Scd1
f/f
) on 4-wk high-fat diet (HFD) developed significantly increased VCAM-1 and Oil Red O positive lesions in the aortic arch, whereas exercise or selective overexpression (OE) of SCD1 in endothelial cells by pAd5/F35/CD144 adenoviral vector mitigated the lesions (
p
< 0.05, n=5, HFD + exercise or HFD+Ad-SCD1 vs. HFD). Arterial stiffness as measured by Pulse Wave Velocity of the left common carotid was elevated in LDLR
-/-
SCD1
EC-KO
on HF diet, but was decreased with exercise or SCD1 OE.

Conclusion:
PPARγ-SCD1 signaling is flow-responsive to mitigate arterial inflammation and stiffness. Despite SCD1-associated cancer cell metabolism, endothelial-specific SCD1 confers vascular protection, consistent with the reported skeletal muscle-specific model. Thus, we demonstrate that exercise activates PPARγ-SCD1 and SCD1 catalyzes AZA and OA as biomarkers with therapeutic potential.
Abstract P3009: Fatty Acid-binding Proteins Promote Pulmonary Arterial Hypertension Via Upregulation Of Endothelial Glycolysis
Authors: LIU BIN , Yi Dan , Pan Jiakai , Ramirez Karina , Fallon Michael , Dai Zhiyu
Published: November 2022

Introduction:
Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients.

Hypothesis:
We hypothesize that endothelial fatty acid metabolism is critical for obstructive vascular remodeling in the pathogenesis of PAH.

Methods:
A severe mouse model of PH

Egln1
Tie2Cre

mice were bred with

Fabp45
-/-

mice to generate

Egln1
Tie2Cre

/

Fabp45
-/-

mice. We applied single-cell RNA sequencing (scRNA-seq) to profile the pulmonary cells in

Egln1
Tie2Cre

mice and

Egln1
Tie2Cre

/

Fabp45
-/-

mice. Human hPAEC from idiopathic PAH patients and healthy donors were used to measure fatty acid-binding protein 4 and 5 (FABP4 and FABP5) expression. siRNA-mediated knockdown of FABP4 and FABP5 and lentivirus-mediated FABP4 and 5 overexpression were performed to study cell proliferation, apoptosis, and glycolysis.

Results:
scRNA-seq analysis demonstrated that both FABP4 and 5 were highly induced in the ECs of

Egln1
Tie2Cre

mice. PAECs from IPAH patients also showed higher expression of FABP4 and 5. Knockdown of FABP4-5 reduced EC proliferation, starvation-induced Caspase 3/7 activity, and glycolysis. Overexpression of FABP4-5 promoted EC glycolysis and proliferation. Genetic deletion of
Fabp4
and 5 in

Egln1
Tie2Cre

mice exhibited a reduction of RVSP, RV hypertrophy, and reduction of EC glycolysis gene programming compared to

Egln1
Tie2Cre

mice.

Conclusions:
FABP4 and 5 control EC glycolysis and contribute to the development of PAH.
Abstract P3013: MicroRNA-122 Targeting With Gamma Peptide Nucleic Acid Inhibitor Improves Vascular Endothelial Dysfunction In Pre-diabetic Mice.
Authors: Gaddam Ravinder R , Dhuri Karishma , Kim Yung-Rae , Jacobs Julia S , Kumar Vikas , Li Qiuxia , Irani Kaikobad , Bahal Raman , Vikram Ajit
Published: November 2022

Background:
In patients with diabetes, the endothelial function precedes and predicts the risk of cardiovascular disorders. Current treatment approaches fail to decrease unwanted cardiovascular outcomes in these patients. The blood levels of microRNA-122 (miR-122) elevated in patients with diabetes and correlated with risk and severity of cardiovascular disorders. Therefore, targeting miR-122 with efficient and safer inhibitors could be a promising approach. The objective of this study was to investigate the effectiveness of gamma peptide nucleic acid (γPNA)-based miR-122 inhibitor in preventing the development of endothelial dysfunction in pre-diabetic mice.

Methods:
To test the effectiveness of γPNA technology in inhibiting miR-122, we synthesized γPNA-miR-122 inhibitor (γP-122-I) and γPNA-scrambled control (γP-SC). The purity and molecular weight of γP-122-I and γP-SC were assessed by HPLC and MALDI spectrometry, respectively. The binding and affinity of γP-122-I were determined by gel-shift assay and thermal denaturation. The insulin-resistance (induced by high-fat-diet, HFD) mice were injected with γP-122-I (5 mg/kg/day, i.p., 6 weeks). miR-122 expression was measured by real-time PCR. The safety of γP-122-I was evaluated by biochemical and histological analysis of vital organs (liver & kidney). The vascular endothelial function was assessed by determining endothelium-dependent and independent relaxation of phenylephrine-induced precontracted aortic rings. Results were expressed as mean ± s.e.m.

Results:
The HPLC of γPNA oligomers show that both γP-122-I and γP-SC were >95% pure. The molecular weight of γP-122-I (m/z: 9227.4) and γP-SC (m/z: 9232.7) were confirmed by MALDI spectrometry. γP-122-I strongly binds with miR-122 and forms a stable complex compared to commercially available miR-122 inhibitor. A comprehensive safety analysis of γP-122-I in mice shows that neither it affected liver and kidney function nor their morphology during acute and chronic exposure, respectively. Next, HFD-fed mice show higher serum and aortic miR-122 levels, endothelial dysfunction, and inflammation, which were reversed by γP-122-I treatment.

Conclusions:
γP-122-I inhibits the development of impaired endothelial function in pre-diabetic mice without any evidence of toxicity
Abstract P3016: Role Of AMPK/Sirtuin3 Signaling In HFpEF-associated Atrial Myopathy
Authors: Tong Dan , Schiattarella Gabriele G , Jiang Nan , Daou Daniel , Luo Yuxuan , Link Mark S , Lavandero Sergio , Gillette Thomas G , Hill Joseph A
Published: November 2022

Background:
Patients with heart failure with preserved ejection fraction (HFpEF) are uniquely predisposed to atrial fibrillation (AF), which significantly worsens clinical outcomes. Pathological atrial remodeling, i.e. atrial myopathy, usually precedes clinical AF and is deemed to be an underlying cause of AF. However, little is known about its molecular features, and no effective treatments have been identified.

Method:
Wild type C57Bl6 mice were fed a high fat diet (HFD) and received L-NAME via drinking water (5-8 weeks). Atrial morphology and function were assessed by echocardiography. AF was induced by transesophageal pacing.

Results:
We discovered that HFpEF mice manifest prominent sinoatrial node (SAN) dysfunction and are highly susceptible to pacing-induced AF, suggesting that this is an ideal model for studying HFpEF-associated atrial changes. Our findings revealed that atrial hypertrophy, contractile dysfunction, and conduction abnormalities are key features of HFpEF-associated atrial myopathy. Interestingly, we did not observe significant atrial fibrosis in this model, a prominent feature of other AF models. Importantly, we discovered impaired AMPK/Sirt3 signaling in the atria of HFpEF mice, and similar atrial defects were observed in mice with cardiomyocyte-specific loss of sirtuin3 (Sirt3) or AMPK, indicating a critical role of AMPK/Sirt3 signaling in HFpEF-associated atrial myopathy. Lastly, we found that metformin, a widely used clinical AMPK agonist, significantly attenuated AF inducibility and SAN dysfunction in HFpEF mice.

Conclusion:
Our “two-hit” HFpEF model successfully recapitulates HFpEF-associated atrial phenotypes. Using this model, we unveiled molecular features of a distinct atrial myopathy. We identified a critical role of AMPK/Sirt3 signaling in HFpEF-associated atrial remodeling and demonstrated the therapeutic effect of metformin. As next steps, confirming this benefit in a clinical trial is warranted.
Abstract P3025: Prevalence Of Distinct Regional Wall Motion Abnormalities Among STEMI Patients Of North Indian Origin
Authors: S. Mohd Shiraz Rizvi , Sunny Sini , Wani Irshad , Mahdi Farzana , Rajasekaran Namakkal-Soorappan
Published: November 2022

Background:
Causal mechanisms and specific consequences of heart failure (HF) with preserved ejection fraction (HFpEF) are unknown. We compared the EF of ST-elevation myocardial infarction (STEMI) patients with the regional wall motion abnormalities (RWMA).

Methods:
2D ECHO data in STEMI patients (n=217) was analyzed for HF with reduced systolic dysfunction (HFrEF), HFpEF, and RWMA.
Inclusion:
Patients with ≥50% stenosis and hypertension.
Exclusion: S
epticemia and malignancy patients.

Results:
Among 217 STEMI subjects, n=102 had either preserved or significantly increased ejection fraction (45-65%), indicating that >47% of them were devoid of systolic dysfunction. The other two major groups were the subjects with moderately abnormal systolic dysfunction (HF
r
EF; EF >31-42%) and severely abnormal dysfunction (HF
r
EF; EF <30%). Over 50% of the subjects with HF
p
EF had basal-septum and inferior wall motion abnormalities, while the rest had apical septum, LAD territory and posterior wall impairments. Four patients displayed signs of mid-septal and anterior wall issues. While most of the HFrEF patients displayed wall motion defects in multiple regions, <10 patients showed anterior wall motion abnormality. Severe HFrEF subjects (n=12) displayed global (all regions) motion defects, which contributes to end-stage heart disease. The RWMA seem to be unique for HFrEF and HFpEF among the STEMI patients.

Conclusion:
Results indicate that STEMI patients could be sub-grouped into both HF
r
EF and HF
p
EF. Further investigation into region-specific rate of remodeling, effect of lifestyle, food habits and pharmacological interventions will aid in improving clinical outcomes.
Abstract P3026: The Effects Of Diet Modification On The Progression Of Heart Failure With Preserved Ejection Fraction
Authors: Cho Jae Hyung , Pellot Isabelle , Kim Myung Yoon , Nawaz Asma , Marbán Eduardo , Cingolani Eugenio
Published: November 2022

Introduction:
Hypertension is a major risk factor for the development of heart failure with preserved ejection fraction (HFpEF). A high-salt diet has shown to induce hypertension and HFpEF in Dahl salt-sensitive rats; however, once developed, it is unclear if HFpEF resolves after a return to low-salt diet.

Hypothesis:
Dietary modification to reduce salt content will decrease blood pressure and lead to resolution of HFpEF in Dahl salt-sensitive rats.

Methods:
Dahl salt-sensitive rats (n=12) were fed a high-salt (HS) diet (8% NaCl) from 7 weeks of age to induce HFpEF. Rats fed a normal-salt (NS) diet (0.3% NaCl; n=6) served as controls. The development of HFpEF was verified with an echocardiogram at 14 weeks of age in HS-fed rats. HS diet was then switched to NS diet in a subset of the HFpEF rats (HFpEF s/p NS, n=6) at 16 weeks of age, while the rest of the HFpEF rats continued to consume HS diet (HFpEF s/p HS, n=6). Blood pressure was measured non-invasively using a tail artery cuff. Echocardiography was performed to measure systolic and diastolic function. Programmed electrical stimulation was performed to induce ventricular arrhythmias.

Results:
In rats with HFpEF, a switch to NS diet improved diastolic function (E/E’ ratio 14.9±3.0 in HFpEF s/p NS vs. 25.3±8.2 in HFpEF s/p HS, p=0.045). However, the diet switch did not reduce systolic blood pressure (204±15 mmHg in HFpEF s/p HS vs. 194±21 mmHg in HFpEF s/p NS, p=0.44). Lung congestion was also significantly reduced after NS diet in rats with HFpEF (lung/body weight 0.39±0.03% in HFpEF s/p NS vs. 0.50±0.06% in HFpEF s/p HS, p=0.0040). Ventricular arrhythmia inducibility did not change after diet switch (100% in both HFpEF s/p HS and HFpEF s/p NS rats), nor did the duration of induced ventricular arrhythmias (75±3 beats in HFpEF s/p HS vs. 67±8 beats in HFpEF s/p NS, p=0.0749). QTc intervals did not decrease after diet changes in rats with HFpEF (226±29 ms in HFpEF s/p HS vs. 225±4 ms in HFpEF s/p NS, p=0.095).

Conclusions:
Diet modification improves diastolic function and reduces pulmonary congestion in Dahl salt-sensitive rats with HFpEF, but it did not reduce blood pressure or inducibility of ventricular arrhythmias. Further mechanistic study is warranted to further investigate the effects of diet on HFpEF progression.