bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P3091: Suppression Of Myeloid Yap Antagonizes Adverse Cardiac Remodeling During Pressure Overload Stress
Authors: Francisco Jamie , Del Re Dominic
Published: October 2023

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction and heart failure. While ischemia-induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non-ischemic stress. Recent work has implicated a role for Yes-associated protein (YAP) in modulating inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non-ischemic stress is not described. We hypothesized that YAP mediates a pro-inflammatory response during pressure overload (PO)-induced non-ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicited myeloid YAP activation, and myeloid-specific YAP knockout mice (YAP
F/F
;LysM
Cre
) subjected to PO stress had preserved systolic function, and attenuated pathological remodeling compared to control mice. Inflammatory indicators were also significantly attenuated, while pro-resolving genes including
Vegfa
were enhanced, in the myocardium of myeloid YAP KO mice after PO. Experiments using bone marrow-derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression shifted polarization toward a resolving phenotype. We also observed attenuated NLRP3 inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Studies in RAW264.7 cells demonstrated the effectiveness of pharmacological YAP inhibition using verteporfin to attenuate pro-inflammatory gene expression, and augment VEGFA levels. Finally, we leveraged nanoparticle-mediated delivery of verteporfin and observed attenuated PO-induced pathological remodeling and inflammation compared to DMSO nanoparticle control treatment. These data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and fibrosis during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non-ischemic heart disease.
Abstract P3098: Immune Suppression Ameliorates A Mouse Model Of Lmna-related Cardiomyopathy With Nuclear Envelope Rupture
Authors: En Atsuki , Bogireddi Hanumakumar , Thomas Briana , Stutzman Alexis , Almakki Omar , Ikegami Sachie , Moskowitz Ivan P , Ikegami Kohta
Published: October 2023

Lamin A/C (encoded by the
LMNA
gene) are the components of the nuclear lamina, a protein meshwork providing the structural integrity to the nuclear membrane. Dilated cardiomyopathy (DCM) caused by
LNNA
gene mutations is the second most common familial DCM in adults and associated with severe prognosis and high mortality. Despite decades of investigations into cell autonomous impacts of
LMNA
mutations, LMNA-DCM lacks effective treatments except for heart transplantation. Here, we identify cell non-autonomous effects of
LMNA
mutations and demonstrate the efficacy of targeting immune activities in mouse models of LMNA-DCM. We induced
Lmna
gene deletion specifically in cardiomyocytes in adult mice and tracked molecular, cellular, and physiological alterations in the heart at a high temporal resolution. We identified frequent localized nuclear envelope rupture, with the concomitant exposure of nuclear DNA to the cytoplasm, as the earliest direct consequence of Lamin A/C protein reduction in cardiomyocytes. Surprisingly, nuclear envelope rupture did accompany cardiomyocyte cell death or extensive transcriptional changes or cytosolic DNA sensing pathway activation within the cardiomyocytes. Instead, it coincided with extensive proinflammatory signaling activation within the cardiac macrophage population. This was followed by extensive fibrosis and structural and functional alterations of the heart, culminating in DCM and death. Treatment of LMNA-DCM mice with the immunosuppressant Dexamethasone suppressed fibrosis, profoundly improved the cardiac structure and function, and conferred a two-fold lifespan extension after
Lmna
gene deletion. This work suggested that immune activation is the major pathological component of LMNA-DCM and an effective target for treating LMNA-DCM patients.
Abstract P3103: Heart Failure In Menopause: Treatment With Rock Inhibitor And Atorvastatin
Authors: Zapata-Sudo Gisele , Da Silva Jaqueline S , Rocha Bruna , Montagnoli Tadeu , Cunha Rebeca S , Nascimento-Carlos Bianca , Fraga Carlos
Published: October 2023

In advanced age, women tend to develop heart failure with preserved ejection fraction (HFpEF) due to estrogen depletion. The signaling pathway of ROCK is involved in cardiac complications consequent to female aging. Thus, this work investigated the comparative effects of atorvastatin and ROCK inhibitor, fasudil on the treatment of HFpEF induced by oophorectomy (OVX) in spontaneously hypertensive rats (SHR). Protocols were approved by the Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Under anesthesia with ketamine (80 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.), female SHR were submitted to OVX procedure and after 8 wks were randomly orally treated with either vehicle or atorvastatin (40 mg/kg) or fasudil (10 mg/kg) during 4 wks. At the end of treatment, hemodynamic parameters were evaluated using echocardiography and LV catheterization. Cardiac tissues were collected for histologic analysis. Cardiac hypertrophy was reversed by treatment with atorvastatin or fasudil. Increased systolic pressure of LV was observed in SHR-OVX group (149.2 ± 7.9 mmHg), which was partially reduced by fasudil (123.4 ± 7.4 mmHg) and normalized by atorvastatin (94.5 ± 3.9 mmHg). Reduction of the increased diastolic dysfunction observed by echocardiogram was confirmed, since the recovery of the increased LV end diastolic pressure of SHR-OVX (32.1 ± 1.8 mmHg) occurred after treatment with atorvastatin (22.4 ± 9.6 mmHg) and fasudil (11.9 ± 2.4 mmHg). Fibrosis was observed because of the increased collagen deposition in SHR-OVX (6.2 ± 0.1%), with partial reduction with fasudil (4.4 ± 0.3%) and normalization after treatment with atorvastatin (1.9 ± 0.7%). Cell proliferation was evaluated using the p38 marker, where the percentage of nuclei marked was determined in relation to the total, through immunohistochemical technique. An increase in nuclear p38 in the SHR-OVX group was observed (23.2 ± 3.2%), however, the treatment with atorvastatin (12.1 ± 1.7%) and fasudil (5.3 ± 1.3%) reduced the nuclear p38 detection. In conclusion, atorvastatin and fasudil promoted recovery of HFpEF induced by estrogen depletion in SHR. Inhibition of ROCK signalling by atorvastatin could be involved in the reduction of diastolic dysfunction observed in OVX-SHR group.
Abstract P3104: Sex Differences In Vasodilation In Embryonic Coronary Arteries
Authors: Nagasawa Shion , Kodama Masami , Sakamoto Kazuho , Kudo Fujimi , Manabe Ichiro , Kurokawa Junko
Published: October 2023

Sex differences exist in circulatory system and the onset of cardiovascular diseases. These differences have been thought to be due to the vasoprotective effects of estrogen. However, there are also sex differences at the early stages of life, such as the survival rate of extremely preterm infants. The mechanisms in which those sex differences are formed at first remain to be elucidated. Therefore, we analyzed the mechanism of sex differences using fetal mouse hearts. In this study, embryonic day 17.5 mice were used to understand sex differences in cardiovascular system just before they are born. First, for morpho-functional analysis of coronary arteries, hearts were removed from embryonic day 17.5 mice, and angiography was performed using nano-particle ink. The ink was injected from the aorta at a constant pressure to visualize the morphology of coronary arteries. We found a significant sex difference in coronary artery diameters. Male coronary arteries were larger under physiological conditions, on the other hand, female coronary arteries were more dilated in the presence of the NO-donor NOC7 and the ROCK inhibitor Y27632. In addition, the same tendency was observed in sections of mouse hearts at embryonic day 17.5. These findings suggest that female’s coronary arteries are more contractile than males. Next, embryonic day 17.5 mouse hearts were dissociated, then sorted with FACS to obtain endothelial cells for bulk RNA-seq and single-cell RNA-seq. Cells were clustered based on the expression of 8 sex-differential genes to identify the sex of each cell. In line with the result of coronary visualization, Gene Set Enrichment Analysis showed the gene set of smooth muscle cell contraction was upregulated in males. Furthermore, cell-cell communication analysis showed that male endothelin signals were modestly stronger in endothelial cells and smooth muscle cells. These data support our previous observation that male left coronary arteries have a larger diameter but are less reactive to vasodilators, because they may have greater contractile function. In conclusion, this study suggests that functional and morphological sex differences already exist at embryonic day 17.5.
Abstract P3107: Sexual Dimorphism In Diastolic Dysfunction And Cardiac Small Vessel Disease In Heart Failure With Preserved Ejection Fraction Mouse Models
Authors: Cornuault Lauriane , Grouthier Virginie , Rouault Paul , Bourguignon Célia , Foussard Ninon , Chapouly Candice , Gadeau Alain-Pierre , Couffinhal Thierry , Renault Marie-Ange
Published: October 2023

Introduction:
Coronary microvascular disease has been proposed to be responsible for heart failure with preserved ejection fraction (HFpEF) about 10 years ago. However, to date the role and phenotype of the coronary microvasculature has still been poorly investigated in animal models of HFpEF and sex differences are not always considered.

Objective:
To characterize the phenotype of the coronary microvasculature in male and female mice with diastolic dysfunction in two HFpEF mouse models.

Method:
We assessed cardiac function and characterized the coronary microvasculature in mice fed with a high fat diet (HFD) + L-NAME regimen and in Leptin receptor deficient (

Lepr
db/db
)

mice. Notably, our study was done in males, females and ovariectomized (OVX) female mice in order to search for possible sexual dimorphisms.

Results:
Upon a HFD + L-NAME regimen, both males and OVX females but not non OVX females develop diastolic dysfunction attested by an increased end diastolic pressure. In Lepr
db/db
mice, both Male and non OVX female mice develop diastolic dysfunction. Notably female mice have reduced estradiol and progesterone level mice mimicking ovariectomy. We found that both OVX and non OVX females but not males display increased endothelial activation attested by increased ICAM1 expression, endothelium leakage attested by increased Fibrinogen and IgG extravasation and decreased arteriole diameter suggesting vasoconstriction. The same results were found in

Lepr
db/db

mice.

Conclusion:
Diastolic dysfunction is not always associated with cardiac small vessel disease since

Lepr
db/db

males and C57BL/6J males fed with a high fat diet (HFD) + L-NAME regimen develop diastolic dysfunction in the absence of endothelial dysfunction. Also endothelial dysfunction may not be sufficient to induce diastolic dysfunction since non OVX female mice fed with a high fat diet (HFD) + L-NAME regimen display endothelial dysfunction while they do not develop diastolic dysfunction.
Abstract P3112: The Effects Of Sex Hormones On Intestinal Lipoprotein Size
Authors: Nauli Andromeda M , Phan Ann
Published: October 2023

Larger intestinal lipoproteins are more likely to be retained in the intestinal wall, allowing their fat to be hydrolyzed and taken up by the abdominal viscera. Since men generally accumulate more abdominal visceral fat than women, we hypothesize that testosterone stimulates the release of larger intestinal lipoproteins relative to estrogen and progesterone. To test our hypothesis, the differentiated Caco-2 cells (n ≥ 12 per group) were induced to produce intestinal lipoproteins while simultaneously subjected to the physiological concentrations of sex hormones by using our semipermeable membrane system. The lipoproteins were isolated by using density gradient ultracentrifugation, and the amount of ApoB and TG were determined. Since each lipoprotein consists of only one ApoB, higher ratios of TG/ApoB suggest larger lipoproteins as shown by our previous studies. Our results indicated that while there was no dose-dependent effect of beta-estradiol (p > 0.05) and progesterone (p > 0.05), testosterone significantly (p = 0.004) increased the TG/ApoB ratio. When these hormones were combined to resemble the plasma concentrations observed in men and the different phases of the ovarian cycle (follicular, ovulatory, and luteal), both the male (p = 0.045) and the luteal phase (p = 0.043) had significantly higher TG/ApoB ratios than the ovulatory phase. The ovulatory phase secreted significantly less amount of TG than the other groups, and the follicular phase secreted significantly less amount of TG than the male (p = 0.0003). ApoB was comparable among all these four groups (p > 0.05). In conclusion, unlike beta-estradiol and progesterone, testosterone significantly increased the size of intestinal lipoproteins. The ovulatory phase secreted the least amount of TG and produced significantly smaller intestinal lipoproteins than the male and the luteal phase. These studies support our hypothesis that through their testosterone effects, men produce larger intestinal lipoproteins. Larger lipoproteins tend to stay longer in the intestinal wall, allowing their fat to be taken up preferentially by the abdominal viscera. Our studies may partly explain why men are more likely to accumulate abdominal visceral fat, which is an independent predictor of mortality.
Abstract P3121: Overcoming Gender Bias In Cardiovascular Research Using Mice Models By Monitoring Metabolic Differences Between Genders Using Indirect Calorimetry
Authors: Kimura Yuka , Gupta Manish K
Published: October 2023

Background:
Metabolic differences between genders have been observed in humans, with studies reporting differences in body composition, metabolic rates, and response to dietary interventions and how they may affect the risk of cardiovascular disease (CVD) and diabetes. However, most research in CVD models has been conducted exclusively on male mice, resulting in strong gender bias. Moreover, in CVD research investigating differences in hypertension, it is important to note the sexual dimorphisms for these pathologies that result from genetic and hormonal differences. More specifically, age-related CVD rates are lower for women, which has been associated with the protective role of sex steroid hormones in women, which affect the prevalence of metabolic syndrome. However, further research is required to investigate the exact role of these hormones in explaining the lower age-specific CVD rates and the metabolic differences between males and females.

Methodology and Result:
Adult wild-type C57B6 mice were housed individually in a metabolic cage. We used the indirect calorimetric method to monitor oxygen consumption, carbon dioxide release, respiratory exchange ratio (RER), and energy expenditure of individual mice. Our initial study shows female mice have better volunteer exercise capacity than male mice.

Conclusion:
This study will address a significant gap in the literature by investigating metabolic differences between genders without gender bias. Our study's findings will provide a foundation for future research in CVD and contribute to developing gender-specific interventions for metabolic disorders.
Abstract P3124: Chronic Infusion Of Angiotensin II Promotes Ventricular Fibrosis And Diastolic Dysfunction In Aging Male But Not Female Mouse Hearts
Authors: Mishra Manish , Khuong Ninh , Banga Shubham , Howlett Susan E
Published: October 2023

Heart failure prevalence increases with age and affects men and women differently. Chronic activation of the renin-angiotensin system (RAS) in heart failure increases circulating angiotensin II (AII) levels, which elevates blood pressure (BP), promotes fibrosis and leads to adverse remodelling of the heart. Whether maladaptive responses to AII differ between the sexes at older ages is unclear, as most studies use young male animal models. We investigated whether adverse cardiac remodeling induced by chronic exposure to AII differed between the sexes in older C57BL/6 mice. Osmotic minipumps with pressor doses of AII (3 mg/kg/day) or saline were implanted into older mice (≈16 mos) of both sexes for 6 weeks. BP (tail cuff) and ventricular structure/function (echocardiography) were measured at baseline, midpoint, and endpoint. Ventricular fibrosis markers (transforming growth factor-ß (TGF-ß), fibronectin collagen I, collagen III) were quantified with qPCR. Mean arterial pressure increased similarly in both sexes (76.3±4.2 vs. 136.1±4.6 mmHg in males and 73.9±2.9 vs. 116.1±8.7 mmHg in females; p<0.05; n=6-8/group). AII infusion also increased LV mass and reduced ejection fraction in males and females. By contrast, AII increased signs of diastolic dysfunction in males but had little impact on diastolic function in females. For example, isovolumic relaxation time increased over 6 weeks of AII infusion in males (11.7±1.0 vs. 19.0±1.4 ms; p<0.05) but not in females (14.6±0.9 vs. 16.4±1.2 ms). Similar results were seen for E/A ratios. Molecular studies showed a marked increase in mRNA abundance for TGF-ß in male ventricles (1.02±0.13 vs. 1.50±0.16; p<0.05) but not in females (1.01±0.08 vs. 1.25±0.10). Similar male-female differences were seen for collagens I and III (fibronectin was similar in both sexes, and not affected by AII). As TGF-ß signalling increases the production of profibrotic mediators like collagens I and III, these findings suggest that chronic RAS activation increases fibrosis and disrupts diastolic function in aging male hearts. By contrast, older female hearts are resistant to these deleterious effects of RAS activation. These sex-specific differences may modify heart failure expression and responses to treatment in older men and women.
Abstract P3133: Role Of Myofilament-associatedNeuronal NOSFor Ca
2+
Desensitization Via Troponin I Phosphorylation In The Right Ventricular Cardiomyocytes Of Rats
Authors: Jeon Young Keul , Kwon Jae Won , Kim Sung Joon
Published: October 2023

This study aimed to compare the role of neuronal nitric oxide synthase (nNOS, NOS1) in the excitation-contraction coupling and myofilament Ca
2+
-sensitivity between left ventricular cardiomyocytes (LVCMs) and right ventricular cardiomyocytes (RVCMs). Previous research had only investigated the effects of nNOS on LVCMs, but our study focused on RVCMs which have slightly larger diastolic sarcomere length (SL
D
) and smaller sarcomere shortening (ΔSL) than LVCMs. Immunoblot study showed that the total expression and phosphorylated Ser
1417
ratio of nNOS were not different between RVCMs and LVCMs. However, treating RVCMs with the nNOS-specific inhibitor, S-methyl-L-thiocitrulline (SMTC), resulted in more prominent augmentation of ΔSL and shortened SL
D
in RVCMs, along with a paradoxical decrease of the amplitude of Ca
2+
transient (Δ[Ca
2+
]
i
) due to a more significant inhibition of L-type Ca
2+
current in RVCMs. The Ca
2+
-contraction loop showed a more leftward shift in the RVCMs by nNOS inhibition, indicating Ca
2+
desensitization of the myofilaments by the intrinsic nNOS. Previous studies offered troponin I (cTnI) phosphorylation as the regulatory target of myofilament Ca
2+
-sensitivity through the sGC-cGMP-PKG pathway downstream to NO. The SMTC treatment decreased cTnI phosphorylation in the RVCMs, while not in the LVCMs, suggesting that nNOS plays a more prominent role in myofilament Ca
2+
desensitization in RVCMs by targeting cTnI. Additionally, the higher phosphorylation of nNOSβ, the isoform of nNOS expressed in the myofilaments, was detected in the myofilament of RVCMs than LVCMs. This study is the first to demonstrate the more significant role of nNOS in RVCMs in regulating myofilament Ca
2+
desensitization through cTnI.
Abstract P3157: MicroRNA-16 Exerts Anti-fibrotic Effects Via Suppression Of Tgfbr3 In Rats With Heart Failure And Preserved Ejection Fraction
Authors: Cho Jae Hyung , Rajewski Alex , Bresee Catherine , Sanchez Lizbeth , Kim Myung Yoon , Mesquita Thassio , Jones Xaviar , Pellot Isabelle , Liu Weixin , Nawaz Asma , Ibrahim Ahmed G , Rogers Russell , Cingolani Eugenio , Marban Eduardo
Published: October 2023

Background:
Cardiosphere-derived cells (CDCs) reverse diastolic dysfunction by attenuating ventricular fibrosis in rats with heart failure and preserved ejection fraction (HFpEF). MicroRNAs packaged in exosomes secreted from CDCs (CDCexo), are discovered to mediate the beneficial effects of CDCs.

Hypothesis:
The anti-fibrotic effects of CDCs are mediated by microRNAs contained in CDCexo.

Methods:
Dahl salt-sensitive rats were fed either a high-salt diet to induce HFpEF, or a normal-salt diet to serve as controls. Echocardiogram-verified HFpEF rats were randomized to receive human CDCexo (n=37) vs. vehicle (n=47) via tail vein weekly for 4 weeks. Control rats (n=16) served as the normal phenotype. RNA sequencing of CDCexo was performed to search for candidate microRNAs. Single-cell RNA sequencing was performed in isolated ventricular cells to investigate the mechanisms of CDCexo. Five most abundant microRNAs were selected and injected via tail vein to HFpEF rats. MicroRNA-injected HFpEF rat hearts underwent single-cell RNA sequencing to investigate its mechanisms.

Results:
HFpEF rats injected with CDCexo demonstrated improved diastolic function compared to HFpEF rats injected with vehicle (E/E’ ratio 12.1±2.6 vs. 18.7±4.6 in vehicle, p<0.0001). Masson’s trichrome staining revealed reduced ventricular fibrosis in CDCexo rats (3.5±1.4 vs. 5.4±1.4 % in vehicle rats, p=0.016). Negative regulation of transforming growth factor beta (TGF-β) receptor signaling pathway (p=0.002) was identified in isolated cardiac fibroblasts after CDCexo injection. MicroRNA-16 was revealed as the most abundant microRNA in CDCexo, and intravenous delivery of microRNA-16 recapitulated the beneficial responses (improvement of diastolic function and attenuation of ventricular fibrosis) of CDCexo in rats with HFpEF. Single-cell RNA sequencing of microRNA-16-injected HFpEF hearts confirmed down-regulation of TGF-β receptor activity (p=0.018) in cardiac fibroblasts via suppression of Tgfbr3.

Conclusion:
CDCexo improved diastolic function by suppressing ventricular fibrosis in rats with HFpEF. MicroRNA-16, the most abundant microRNA in CDCexo, recapitulated the beneficial responses of CDCexo in HFpEF rats, via suppression of Tgfbr3 in cardiac fibroblasts.
Abstract P3162: Evaluating Cargo And Cardioprotective Effects Of Extracellular Vesicles From Human Right Atrial Appendage Mesenchymal Cells
Authors: Czosseck Andreas , Chen Max M , Hsu Chuan-Chih , Meeson Annette , Oldershaw Rachel , Lundy David
Published: October 2023

Our team has previously shown the isolation and culture of a mesenchymal cell population from the right atrial appendage (RAA) of living human donor tissues. The cells are CD44/105/166
+
, CD45/19/11b
-
and secrete growth factors (including VEGF and IGF-1) comparable to BM-MSCs. Their secretome preserved EF% and dP/dTmax in a mouse MI model and stimulated angiogenesis in a mouse limb ischemia model.

In the current study, we specifically investigated using these cell-derived extracellular vesicles (EV) as a therapeutic. First, we validated our isolation technique and profiled EV miRNA cargo using PCR arrays. We then assessed the ability of EVs to protect human cardiomyocytes in a hypoxic (1% O2) hiPSC-CM model. Lastly, we used RNA-seq to compare the transcriptome of EV-treated hiPSC-CMs against hypoxic and normoxic controls.

Results confirmed isolation of high purity EVs, showing (by cryoEM) ~120 nm bilayer membrane-bound vesicles, positive for CD81, CD63, ALIX, TSG101 and other EV markers (by antibody array). In the EV cargo we identified 363 known miRNAs present in 3 donor cell isolates, including hsa-miR-21-5p, -202-5p, -1260a and -125b. Target prediction of the most abundant miRNAs ranked GO:0010667 “negative regulation of cardiac muscle cell apoptotic process” as highly significant (26.3%, P = 1.6 x10
-8
), as well as other predicted pathways related to angiogenesis, cardiac development and regulation of inflammation.

A single, transient, treatment of donor EVs (66 ng EV protein/μl) reduced LDH leakage from hypoxic hiPSC-CMs, preserved morphology, and increased viability at 48 hrs and 7 days after injury. RNA-seq of the EV-treated hiPSC-CMs revealed strong upregulation of pathways related to PI3k-akt, calcium signaling and ECM binding compared to hypoxic hiPSC-CMs with vehicle control. We are currently validating key RNA-seq results.

In conclusion, we analyzed and confirmed cardioprotective properties of EVs derived from human RAA samples using a human model system. Combined with our published animal studies, these data indicate that this cell population is a clinically-relevant source of donor cells for cardiac cell therapy. The EVs are also an attractive cell-free potential therapeutic option.
Abstract P3174: Loss Of Hmgcs2-mediated Ketogenesis Accelerates Cardiac Aging
Authors: Lee Yi-Chan , Cheng Yuan-Yuan , Chien Hsin-Yuan , Ruan Shu-Chian , Lin Po-Ju , Hsieh Patrick C
Published: October 2023

Introduction:
We have previously reported that the ketogenic enzyme HMGCS2 regulates metabolic changes to ameliorate cardiac damage by promoting adult cardiomyocyte dedifferentiation and proliferation after myocardial infarction (Cheng et al.
Circulation
, 2022). Intriguingly, ketogenic diet has been shown to prolong the lifespan of aged mice. However, whether the loss of HMGCS2 links to cardiac aging remains unclear.

Hypothesis:
We tested if loss of HMGCS2 results in metabolic change and heart dysfunction and leads to premature cardiac aging.

Methods and Results:
Conventional HMGCS2 knock-out (KO) mice were generated by inserting loxP sites flanking exon 2 of HMGCS2 to eliminate HMGCS2 protein production. HMGCS2 expression and β-hydroxybutyrate production were eliminated in the livers and hearts in HMGCS2 KO mice. Echocardiographic analysis and intraventricular catheterization did not display heart functional differences between wild-type and HMGCS2 KO mice at 3-month-old. However, growing with age, HMGCS2 KO mice revealed a declining survival rate, accompanied by worse diastolic and systolic functions including reduced left ventricle ejection fraction, myocardial performance index, fractional shortening, stroke volume, end-diastolic dimension, end-systolic dimension, isovolumic relaxation time, and left ventricular diastolic time constant (Tau) in HMGCS2 KO mice at 12-month-old. Histological analyses also exhibited aging-related phenotypes in the HMGCS2 KO heart at 12-month-old, including cardiac hypertrophy and fibrosis. Furthermore, mitochondrial analyses indicated mitochondrial impairments of function and structure.

Conclusion:
These results suggest that loss of HMGCS2 accelerates cardiac aging with declining cardiac and mitochondrial function.
Abstract P3176: One Carbon Metabolism Defect In Failing Heart
Authors: Jung Jae Woo W , Flam Emily , Bedi Kenneth , Jang Cholsoon , Rabinowitz Joshua D , Margulies Kenneth B , Arany Zoltan
Published: October 2023

Aberrant cardiac metabolism has long been hypothesized to contribute to heart failure (HF). However, understanding of cardiac metabolism in HF remains incomplete. To fill this gap, we performed metabolomic and RNA sequencing analysis of 48 non-failing (NF) and 39 dilated cardiomyopathy (DCM) hearts. Among many differences, metabolites from one-carbon metabolism were significantly reduced, especially methionine (Met) and s-adenosylmethionine (SAM), a universal methyl donor of methylation. We probed the mechanism behind decreased Met/SAM in DCM hearts. Studies in neonatal rat ventricular myocytes (NRVMs), and
in vivo
steady-state infusions with isotope tracing showed that Met was mostly imported, rather than recycled, suggesting a defective Met transport in DCM hearts. We next explored the impact of low SAM. DNA and histone methylation remained unchanged, but creatine level was significantly decreased. So, we tested potential creatine synthesis in the heart that was previously unclear. To our surprise, NRVMs synthesized ~50% of their creatine pool when external supply is low, and synthesis was dependent on SAM availability. In summary, we find that the majority of Met in cardiomyocyte is imported, with rapid turnover. We also find that cardiomyocytes synthesize creatine and increase creatine production under stress. Together, these findings suggest that defective Met import may contribute to low creatine and SAM levels and ultimately HF.
Abstract P3177: Complex I-induced Protein Turnover, A Novel Mitochondrial Protein Quality Regulation Clipt Goes Beyond The Mitochondrial Calcium Uniporter
Authors: Lee Sandra , Eberdhardt David , Balderas Enrique , Balynas Anthony , Yin Xue , Chaudhuri Dipayan
Published: October 2023

In the setting of mitochondrial disease, patients with cardiac involvement have a nearly threefold increased mortality. Yet, patients often survive months to years suggesting that in cardiomyocytes, there is a compensatory mechanism that maintains energy synthesis despite defective mitochondria. We have recently shown that enhanced activity of the mitochondrial Calcium (Ca
2+
) uniporter channel preserves energetic homeostasis in mitochondrial cardiomyopathies. Here, we describe a novel mechanism responsible for this enhancement. During normal physiology, interaction between the uniporter and Complex I of the electron transport chain promotes its degradation. However, upon Complex I dysfunction, loss of this interaction results in increased functional channels. We termed this form of mitochondrial protein regulation, Complex I-Induced protein turnover (CLIPT). We hypothesize that CLIPT is not unique to the uniporter. To identify other candidates subject to CLIPT, we performed a pulse-chase screen using TurboID, a biotin-ligase proximity labeling tool. HEK cells expressing a mitochondrial TurboID were grown with or without Complex I inhibitors. Following a pulse-chase with biotin, proteins were purified with streptavidin and identified through mass spectrometry. Of these proteins, whose lifetimes increased after Complex I inhibition, Peroxiredoxin-3 (PRDX-3) is a mitochondrial antioxidant shown to provide protection against cell death and in myocardial infarction. Using heterologous expression in c2c12 cells, inhibition of Complex I prolongs PRDX-3 lifetime. Thus, our data suggests that CLIPT can affect mitochondrial proteins beyond the uniporter.
Abstract P3179: Tristetraprolin Regulates Metabolic And Epigenetic Remodeling In Cardiomyocytes Post Myocardial Infarction
Authors: Mahmoodzadeh Amir , Akrami Ashley E , Keykhaei Mohammad , Koleini Navid , Ardehali Hossein
Published: October 2023

Myocardial infarction (MI) triggers profound remodeling in the infarcted, border (BZ), and remote zones of the myocardium. Here, we studied the metabolic remodeling and epigenetic modifications that occur in response to inflammation in the BZ, focusing the role of an mRNA-binding protein that causes degradation of its target mRNA molecules, tristetraprolin (TTP). Inflammatory cells were observed to increase in the BZ, associated with the release of IL-1B and IFN-γ. Among proteins that are altered in the BZ cardiomyocytes, TTP displayed a significant increase in its mRNA and protein levels in the BZ. Isotope tracing using U-13C6 glucose in TTP-overexpressing cells showed that TTP overexpression results in an increase in TCA metabolites upstream of alpha ketoglutarate dehydrogenase (αKGDH), and a decrease of downstream metabolites, suggesting blocked activity at the level of αKGDH. We then identified the mRNA of the E2 component of αKGDH, Dihydrolipoamide S-Succinyltransferase (DLST), to be a target of TTP. Consistent with these findings, DLST mRNA and protein levels are decreased in BZ. DLST needs to be lipoylated to become active, and our data indicate that the mRNA for a key enzyme in DLST lipoylation (lipoic acid synthase, LIAS) is also a target of TTP. Overexpression of TTP led to a decrease in the level of DLST lipoylation. Epigenetic studies showed that, overexpression of TTP resulted in significant decrease in acetylation and methylation of H4K20. Finally, we demonstrated that heart specific TTP KO mice are protected against ischemic injury compared to WT mice, indicating that deletion of TTP alters the remodeling of the BZ, through its role in the regulation of αKGDH and production of αKG in response to infiltration of inflammatory cells.
Abstract P3188: Establishing The Mitochondrial Citrate Transporter As A Regulator Of Cardiac Morphogenesis
Authors: Ohanele* Chiemela , Peoples* Jessica , Karlstaedt Anja , Gayle Ashley , Ghazal Nasab , Sohani Fateemaa , Brown Milton E , Davis Michael , Faundez Victor , Kwong Jennifer Q
Published: October 2023

Congenital heart disease (CHD) results from structural and functional defects of the heart that arise during embryonic development. CHD represents the most common type of birth defect and in the United States alone, affects ~1% of births per year. One common genetic cause of CHD is 22q11.2 deletion syndrome (22q11.2DS), where ~75% of 22q11.2DS patients present with CHD.
SLC25A1
, which is found within the 22q11.2DS critical deletion region, encodes for the mitochondrial citrate carrier, a solute transporter localized to the inner mitochondrial membrane. In developing a knockout mouse model to study the in vivo functions of SLC25A1, we uncovered an unexpected perinatal-lethal phenotype that suggests roles for SLC25A1 in heart development. Hearts from
Slc25a1
knockout embryos display a striking array of cardiac malformations including persistence of an expanded zone of trabeculated myocardium, reduced compact myocardium, and ventricular septal defects. Analysis of mitochondrial structure and function reveal that loss of
Slc25a1
causes mitochondrial ultrastructural and functional defects. Lastly, transcriptomics analyses of metabolism-related genes revealed that
Slc25a1
deletion causes widespread alterations in metabolic gene expression in a dosage-dependent manner. Our results highlight a novel role for SLC25A1 in cardiac morphogenesis and the transcriptional control of metabolic networks in the developing heart and point to a new role for SLC25A1 in the metabolic maturation of the developing myocardium.
Abstract P3191: Hur Mediates Calcium-driven Thermogenesis In Brown Adipose Tissue
Authors: Guarnieri Adrienne
Published: October 2023

Since the discovery of brown adipose tissue (BAT) in adult humans, its high energy capacity has driven investigations targeting adipose tissue thermogenesis as a supplement to whole-body energy expenditure to mitigate the energy imbalances observed in obesity. Uncoupling protein 1 (UCP1) is the primary driver of thermogenesis, however several additional UCP1-independent pathways have been identified. We previously reported that adipocyte deletion of the RNA binding protein human antigen R (HuR) results in thermogenic dysfunction independent of UCP1. RNA sequencing revealed the downregulation of several genes involved in calcium ion transport upon HuR deletion. Although recently established as a thermogenic mechanism in beige adipocytes, the effects of calcium-mediated thermogenesis in brown fat and its contribution to whole-body metabolism remain unexplored. Therefore, we set out to investigate the impact and regulatory mechanism of HuR on SR calcium cycling and heat generation in BAT. We hypothesize that HuR regulates thermogenesis in brown adipose tissue through control of RyR-mediated SR calcium release and assessed this experimentally using murine models with a BAT-specific deletion of HuR. Brown adipocytes isolated from BAT-HuR
-/-
mice were imaged for cytosolic calcium and heat using Fluo-4 calcium indicator and ERthermAC dye respectively. Cohorts of mice were also subjected to acute cold challenge or placed on a 12-week high fat diet study to assess whole-body thermal and energy metabolism. We now show that loss of HuR decreases adrenergic-stimulated calcium release and heat generation in brown adipose tissue, and that this effect is rescued by pharmacological stabilization of RyR2. We also report that HuR directly binds RyR2 mRNA
in vivo
and loss of HuR reduces RyR2 mRNA transcript stability. In assessing whole-body metabolism, we observed impaired thermal tolerance of BAT-HuR
-/-
mice from controls, as well as sex-dependent effects on diet-induced weight gain between models. Our findings reveal, for the first time, that brown adipocytes participate in calcium-mediated thermogenesis and identify a regulatory role for HuR in coordinating heat production through maintenance of RyR2 calcium release activity.
Abstract P3192: Prdm16 Regulates Cardiac Metabolic Genes Through Perm1
Authors: Rouzbehani Omid , Goodman Michael , Sun Bo , Ghosh Rajeshwary , Sabri Amira , Warren Junco S , Landstrom Andrew P , Boudina Sihem
Published: October 2023

The heart is a metabolically demanding organ that requires significant amounts of energy to function properly, and even minor metabolic deficiencies during development can lead to heart failure. Cardiomyopathies are a group of conditions that affect the heart's contractile function, and PRDM16 deficiency is a primary cause of cardiomyopathies associated with 1p36 deletion syndrome in humans. Conditional deletion of Prmd16 in the heart causes a spectrum of cardiac phenotypes including left ventricular non-compaction, late-onset heart failure, and dilated cardiomyopathy. Still, the mechanisms underlying these cardiac phenotypes with loss of Prdm16 are not fully understood. We and others demonstrated that lack of Prdm16 in the heart causes alterations in fatty acid (FA) oxidation and oxidative phosphorylation (OXPHOS) gene expression. Further, we now show that Prdm16 cardiac deletion reduces the expression of key regulators of FA oxidation and OXPHOS genes including Perm1. Loss and gain of Prdm16 function in vitro confirmed a cell-autonomous regulation of Perm1 by Prdm16 in H9c2 rat cardiomyoblasts and neonatal rat ventricular myocytes (NRVMs). Prdm16 ChIP-sequencing analysis of embryonic hearts revealed direct binding of Prdm16 to the promoter of Perm1. Prdm16 binding to Perm1 promoter was further confirmed by ChIP-qPCR in H9c2 cells. Knockdown of Prdm16 in NRVMs reduced maximal mitochondrial respiration, which was rescued by over-expressing Perm1. Further, PERM1 mRNA was diminished in human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) derived from a patient with a PRDM16 mutation (Q187X). These results are evidence that PRDM16 is an upstream regulator of PERM1 in the heart.
Abstract P3197: Sarm1 Deficiency Protects Mice Against Diabetic Cardiomyopathy: Potential Mechanisms Via Ameliorating Lipotoxicity
Authors: Minor Keaton
Published: October 2023

Heart failure occurs at nearly twice the rate in diabetic patients as compared to normal patients. NAD levels are decreased in human and murine heart disease models including diabetic hearts. Strategies to raise NAD in these models have primarily focused on increasing NAD synthesis. Alternatively, inhibiting NAD consumption provides another viable path to increasing NAD levels in diabetic hearts. Sterile Alpha and Tir Motif Containing 1 (SARM1) is an NAD hydrolase that mediates axonal degeneration through NAD degradation and promotion of mitochondrial dysfunction. However, the role SARM1 plays in heart disease has not been investigated. We subjected male wild type (WT) and global SARM1 knockout mice (KO) to chronic diabetic stress induced by streptozotocin injections. We showed that 16-week diabetic stress promoted progressive decline in systolic and diastolic function as measured by longitudinal echocardiography. SARM1 deletion (diabetic KO) improved systolic and diastolic function of diabetic mice, despite similar glucose, and plasma aqueous and lipid metabolite levels as the diabetic wild-type mice. Diabetic KO hearts showed increased NAD levels, suggesting that SARM1 may be activated to promote NAD decline in diabetic hearts. Transcriptomic analysis identified 1948 differentially expressed genes in diabetic WT hearts, compared to non-diabetic WT hearts. Gene Module Network Analysis identified upregulation of fatty acid metabolic processes and suppression of genes involved in mitochondrial processes in diabetic hearts. We measured mitochondrial oxygen consumption rate and found suppressed mitochondrial respiration in diabetic WT hearts. SARM1 deficiency reversed the upregulated fatty acid metabolic genes, improved mitochondrial respiration, and elevated NAD levels in diabetic hearts. Genes such as CD36, HADHA, Acot2, and malonyl-CoA are all downregulated by SARM1 deficiency, indicating that SARM1 deficiency may relieve lipotoxicity by improving mitochondrial function and modifying metabolic transcriptional networks in diabetic hearts. Future analysis will shed light on how SARM1 contributes to lipotoxicity as a new pathogenic mechanism of diabetic cardiomyopathy.
Abstract P3201: Fatty Acid Oxidation Decreases Independently Of Up-regulated Glucose Utilization In The Setting Of Elevated Afterload
Authors: Piristine Hande C , May Herman I , Jiang Nan , Olivares-Silva Francisco , Elnwasany Abdallah , Szweda Pamela , Szweda Luke I , Kinter Caroline , Kinter Michael T , Sharma Gaurav , Wen Xiaodong , Malloy Craig , Jessen Michael E , Gillette Thomas G , Hill Joseph A
Published: October 2023

Background:
To support the unremitting contractile function of the heart, free fatty acids (FFA) are the substrate of choice, with a shift toward greater reliance on glucose when increased cardiac output is required. However, in the setting of obesity or diabetes, this metabolic flexibility is impaired. The putative necessity of a shift in substrate utilization has been hypothesized to occur due to the lower oxygen requirement of glucose versus fat metabolism, but this has never been tested conclusively. Furthermore, in the case of increased afterload, whereas oxygen demand increases, myocardial oxygen availability remains adequate for fatty acid oxidation (FAO). Therefore, a “preference” for glucose has been proposed.

Methods and Results:
Pyruvate dehydrogenase complex (PDC) is the rate-limiting enzyme linking glycolysis to the TCA cycle. By over-expressing PDC kinase 4 (PDK4) in cardiomyocytes, we ensured that PDC was phosphorylated and thereby inhibited. This leads to diminished use of pyruvate, limiting energy production primarily to FAO. Unlike high fat diet feeding, which also increases PDK4 and inhibits PDH, this model positioned us to query the impact of PDH inhibition under elevated workload demand in the absence of systemic high fat conditions. As expected, PDK4 transgenic mice manifested normal cardiac output at baseline. However, they manifested a rapid and severe decline in contractile function when challenged with increased afterload triggered by either L-NAME or surgical transverse aortic constriction (TAC). Metabolic flux analysis revealed that, after TAC, fractional FAO decreased - surprisingly - independent of an increase in glucose/pyruvate utilization. Additionally, proteins involved in the transport and oxidation of FFA were paradoxically down-regulated after TAC regardless of genotype.

Conclusions:
These data demonstrate that cardiomyocytes in a setting in which glucose utilization is clamped and prevented from increasing do not compensate for the deficit in glucose utilization by up-regulating FFA use. In aggregate, our findings suggest that reliance on glucose in the setting of pathological cardiac stress is largely a necessity rather than a simple preference.
Abstract P3205: Differential Adaptation To Mitochondrial Ca
2+
Overload In The Left And Right Ventricles
Authors: Sung Jae Hwi , Chapoy Villanueva Hector , Prins Kurt W , Liu Julia C
Published: October 2023

Mitochondrial Ca
2+
(mtCa
2+
) overload has emerged as a potential contributor to heart failure (HF), possibly through inducing oxidative stress. In HF, right ventricular (RV) function is predictive of mortality, and the RV is more vulnerable to oxidative stress than the left ventricle (LV). Therefore, how the response to mtCa
2+
overload might differ in the RV and LV is critical; however, ventricle-specific mtCa
2+
handling is still poorly understood. We sought to induce cardiac mtCa
2+
overload via genetic manipulation of the mitochondrial calcium uniporter complex (mtCU) formed by the MCU pore and several regulatory subunits. mtCU stoichiometry shapes mtCa
2+
kinetics: loss of mitochondrial calcium uptake 1 (MICU1) induces mtCa
2+
overload, and reduced essential MCU regulator (EMRE) to MCU ratio decreases mtCa
2+
uptake rate. Hypothesizing that mtCa
2+
overload differentially affects LV and RV function, we generated tamoxifen (Tmx)-inducible cardiac-specific
Micu1
knockout mice (

Micu1
cKO
)

. After confirming that MICU1 loss at 1 week post-Tmx resulted in mtCa
2+
overload in both ventricles, we explored how the LV and RV responses diverge. In

Micu1
cKO

mice, LV function assessed by echocardiography declined at 4 weeks post-Tmx but recovered partially by 6 weeks. However, impaired RV function at 4 weeks post-Tmx persisted at 6 weeks and longer, without evidence of pulmonary hypertension. Interestingly, at 7-9 weeks post-Tmx, mtCa
2+
levels and oxidative stress remained elevated in the RV but returned to control levels in the LV. Concurrently, EMRE protein levels and mtCa
2+
uptake rate were reduced only in the LV, suggesting that the LV has greater capacity to adapt to mtCa
2+
overload via downregulation of EMRE. We further traced this LV-specific adaptive response to increased maturation of the mAAA-proteases that degrade EMRE. In summary, mtCa
2+
overload induced by MICU1 deletion results in greater oxidative stress and functional impairment over time in the RV than the LV due to absence of compensatory EMRE reduction in the RV.
Fables (Qiṣaṣ) and Muslim Cultural Discourse in Nigeria
Authors: Ibrahim Musa
Published: October 2023

Qiṣaṣ, historical and moral stories told in the Quran and hadiths, are among the factors that make and reshape Muslim cultural discourse. While Quranic sources are short and often not elaborated enough to provide a context from which different scenarios could be created, Muslims rely on fables from contested sources to adapt the qiṣaṣ to various cultural environments, languages, and media. In this context, qiṣaṣ become influential not only because of the power of storytelling, but how their religious contents could be appropriated or even manipulated to suit local narratives and be used as powerful discursive tools within Muslim culture. Using aṣḥāb al kahf (the People of the Cave), a story told in the Quran, as a case study, this article looks beyond mere changes that emerged in the process of adaptations and circulations via various media (especially film) to analyzing how the changes are embedded within local Muslim discourse involving different actors and reform movements in Nigeria.
Taxonomic and nomenclatural notes on Brazilian Dichorisandra (Commelinaceae)
Authors: AONA LIDYANNE YURIKO SALEME , BITTRICH VOLKER , AMARAL MARIA DO CARMO E.
Published: October 2023

Based on a revision of Dichorisandra (Commelinaceae), this work proposes new synonyms and designates types for 14 species that occur in Brazil. Comments on the taxonomy, nomenclature, and the distribution and habitats occupied by each of these species are provided.