bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Tu095: Cycloastragenol Alleviates Cardiac Fibrosis through Inhibiting Pyroptosis Activated by NLRP3 Inflammasome
Authors: QI RONG
Published: October 2024

Cardiac fibrosis is cardiac interstitial remodeling characterized by an enhanced extracellular matrix deposition. Acute sympathetic stress causes cardiac cells to immediately undergo pyroptosis, which induces cardiac inflammation and damage, and ultimately results in cardiac fibrosis. Our previous study has demonstrated the inhibitory effects of cycloastragenol (CAG), an active form of astragaloside IV isolated from Astragalus membranaceus, on isoproterenol (ISO)-induced cardiac fibrosis through blocking the NLRP3 signaling pathway. It is yet unknown how CAG affects pyroptosis of cardiac cells and whether its ability to prevent cardiac fibrosis coincides with this process. In this study, 5 mg/kg ISO was subcutaneously injected into wild-type (WT), NLRP3 knockout (KO), and GSDMD KO mice for 7 days to induce cardiac fibrosis. CAG (125 mg/kg/d) was administrated to the mice intragastrically at the start of the ISO injection. Primary cardiomyocytes and cardiac fibroblasts were isolated from neonatal rat hearts and treated with 10 μM ISO either with or without CAG. The results show that CAG alleviates cardiac fibrosis in WT mice, while lost this efficacy in NLRP3 KO mice. GSDMD KO mice do not develop ISO-induced cardiac fibrosis, indicating the key role of pyroptosis in cardiac fibrosis. Pyroptosis mainly occurs in mouse hearts during the initial stages of ISO induction (1 and 24 hours after ISO injection), and CAG significantly reduces pyroptosis of heart cells in WT mice, while CAG is unable to prevent pyroptosis in NLRP3 KO mouse hearts. The TGF-/Smads signaling pathway is also inhibited by CAG in WT mice but not in NLRP3 KO mice. Additionally, CAG prevents the in vitro pyroptosis of cardiomyocytes induced by ISO directly and cardiac fibroblasts caused by LPS+ATP. These results suggest that the principal mechanism of the anti-cardiac fibrosis effect of CAG mainly depends on its inhibition of pyroptosis mediated by NLRP3 inflammasome.
Abstract Tu104: Mitochondrial Hydrogen Sulfide Regulates Skeletal Muscle Dysfunction and Exercise Intolerance in Cardiometabolic HFpEF
Authors: Allerton Timothy , Doiron Jake , Elbatreek Mahmoud , Li Zhen , Stampley James , Davis Gregory , Noland Robert , Irving Brian , Sharp Thomas , Xia Huijing , Shah Sanjiv , Lefer David
Published: October 2024

Introduction:
Cardiometabolic heart failure with preserved ejection fraction (HFpEF) results in severe exercise intolerance. Dysregulated skeletal muscle metabolism and mitochondrial function are key drivers of exercise intolerance in cardiometabolic HFpEF. Mitochondrial hydrogen sulfide (H
2
S) generated by 3-mercaptopyruvate sulfur transferase (3-MST) exerts potent bioenergetic properties. We have recently shown that H
2
S bioavailability is significantly attenuated in HFpEF. The role of H2S in skeletal muscle metabolism and exercise tolerance in HFpEF is currently unknown.

Hypothesis:
We tested the hypothesis that the loss of skeletal muscle H
2
S results in metabolic dysfunction in skeletal muscle and contributes to exercise intolerance in cardiometabolic HFpEF.

Methods:
Skeletal muscle (gastrocnemius) from L-NAME + High Fat diet (“two-hit”) treated, and chow-fed control male mice (n=10/group) were assessed for free H
2
S and 3-MST enzymatic activity using GC-chemiluminescence detection. Additionally, muscles were processed for RNA sequencing. Next, male global 3-MST knockout (KO) mice (n=12) and controls (n=10) were subjected to “two-hit” HFpEF for 10 weeks, and exercise tolerance was evaluated. Skeletal muscle (soleus and gastroc) was subjected to high-resolution respirometry and substrate oxidation of radiolabeled isotope (
14
C) for leucine and palmitate.

Results:
Skeletal muscle H
2
S levels (0.73 ± 0.18 vs. 1.0 ± 0.24 nmol/g/tissue) and 3-MST activity (0.46 ± 0.01 vs 0.57 ± 0.01 µmol/g/tissue/min) were significantly (p < 0.05) reduced in “two-hit” HFpEF mice compared to control mice. At the transcriptome level, 3-MST was one of the top 50 differentially regulated genes (i.e., reduced expression) in “two-hit” mice (Adjusted p=0.01). Genetic deficiency of 3-MST KO mice exacerbated (p < 0.05) exercise intolerance as measured by treadmill running distance (53 ± 24 vs. 138 ± 104 meters) vs. “two-hit” HFpEF genotype controls. Mitochondrial Complex I (low ADP) respiration was reduced by 23% in 3-MST KO HFpEF mice (p < 0.05). Genetic deficiency of 3-MST also reduced skeletal muscle leucine oxidation (58 ± 19 vs 84 ± 8; p = 0.01) and dramatically increased incomplete palmitate oxidation by 2.5-fold (p < 0.001) without changing complete oxidation.

Conclusion:
We demonstrate that skeletal muscle mitochondrial H
2
S is a critical regulator of metabolic function and exercise capacity in cardiometabolic HFpEF.
Abstract Tu105: Inhibition of Queuine tRNA-Ribosyltransferase 1 Ameliorates Heaptic Lipogenesis and Atherosclerosis
Authors: Wu Runda , zhang shuning , ma Yuanji , Gao Wei , Liao Jianquan , Weng Xinyu , Teng Yongduan , Zhang Lei , Xu Lili , Su Ya , Wang Wanxin , Dai Yuxiang , Hong Shangyu , Yao Kang , Ge Junbo
Published: October 2024

Background:
Atherosclerosis threatens cardiovascular health, which is featured by abnormal lipid metabolism and chronic inflammation in arterial wall. Queuine tRNA-Ribosyltransferase 1 (QTRT1) , is an enzyme responsible for tRNA modification at the wobble base.

Research Questions:
It is unclear whether the function of QTRT1 is involved in metabolism of non-cancer cells and tissues, especially lipid metabolism mainly orchestrated by liver.

Aims:
We aimed to explore the role of QTRT1 deficiency in hyperlipidemia, liver steatosis and atherosclerosis.

Methods:
Transgenic mice such as Qtrt1fl/flAlb-iCre+/- (Qtrt1LKO), Qtrt1fl/fl(WT), ApoE-/-, and gain of function of PCSK9 by adeno-associated virus gene transfer were used to determine the functional significance of QTRT1 in atherosclerosis. Synthesis, transportation and oxidation of lipids in livers and hepatocytes were examined by quantitative real-time polymerase chain reaction. Moreover, primary hepatocytes with overexpression and downregulation of QTRT1 were followed by RNA sequencing to identify downstream targets.

Results:
Deficiency of hepatic QTRT1 significantly attenuated hyperlipidemia, liver steatosis and atherosclerotic burden, but increased plaque stability in the aorta of mice. Depletion of QTRT1 markedly downregulated de novo lipogenesis (DNL) without influence on lipoprotein transportation and fatty acid oxidation. Qtrt1LKO mice on a high-carbohydrate diet displayed relatively reduced hyperlipidemia and alleviation of DNL. RNA-seq indicated that odorant binding protein 2A (OBP2A) was upregulated in Qtrt1LKO hepatocytes, which reversed the promotion of DNL by QTRT1 in hepatocytes.

Conclusion:
Inhibition of QTRT1 in hepatocytes ameliorates hepatic lipogenesis and atherosclerosis in mice. Targeting of QTRT1 is a promising strategy to improve therapeutic effectiveness in both hyperlipidemia and atherosclerosis.
Abstract Tu106: Cardiomyocyte knockout of Ceramide Synthase 5 protects against metabolic cardiomyopathy and obesity in mice
Authors: Kovilakath Anna , Dail Jordan , Mauro Adolfo , Valentine Yolander , Jamil Maryam , Subler Mark , Windle Jolene , Kronke Martin , Salloum Fadi , Cowart Lauren
Published: October 2024

Introduction:
The prevalence of metabolic cardiomyopathy in the absence of hypertension is on the rise. Ceramide Synthase 5 (CerS5) has been implicated in the pathophysiology of this cardiomyopathy.

Hypothesis:
Depletion of cardiomyocyte CerS5 protects against metabolic cardiomyopathy.

Methods:
Inducible cardiomyocyte-specific CerS5 knockout (CerS5 cKO) mice were generated and placed on a milk-fat diet (HFD) or control diet (CD) for 18 weeks along with control (Myh6-MCM) mice (n=6-11/group). Diet-induced metabolic syndrome was monitored through weekly weight measurements, glucose and insulin tolerance tests, and blood plasma collection for Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). Echocardiography and systemic blood pressure were analyzed at baseline and end of diet. Hearts were assessed for hypertrophy, targeted lipidomics, untargeted metabolomics, and markers associated with cardiometabolic pathophysiology.

Results:
CerS5 cKO mice on HFD exhibited protection from increased heart mass (Fig. 1A-B, KO:6.1±0.2 vs. control:7.6±0.3 mg/mm, P=0.0001), cardiomyocyte hypertrophy (Fig. 1C, KO:252.6±8.1 vs. control:315.2±14.0 µm
2
, P=0.0084), and reduced ejection fraction (Fig. 1D, KO:65.8±3.0 vs. control:51.7±2.4 %, P=0.0047) compared to controls on HFD. Systemic systolic (SBP) and diastolic (DBP) blood pressures at 18 weeks on diet did not vary between groups (Fig. 1E-F). KO mice on HFD also showed protection from metabolic comorbidities, including obesity (Fig. 2A-B, KO:26.1±1.1 vs. control:30.7±1.7 g, P=0.0192), increased HOMA-IR (Fig. 2C-D, KO:12.6±3.0 vs. control:26.0±3.4, P=0.0015), reduced glucose and insulin insensitivity (Fig. 2E-L) compared to control on HFD. This phenotype was associated with reduced CerS5-derived sphingomyelin C14 (Fig. 3A, KO:8.9±1.4 vs. control:164.5±13.5 pmol/mg protein, P=0.0082), C16 (Fig. 3A, KO:168.0±16.8 vs. control:812.4±287.6 pmol/mg protein, P<0.0001) and ceramide C16 (Fig. 3B, KO:34.6±6.2 vs. control:112.7±16.1 pmol/mg protein, P<0.0001), in the heart. Excessive autophagy (Fig. 3C-D) was observed in the hearts of control mice on HFD, but not in the KO mice on HFD. The heart metabolome of CerS5 cKO mice on HFD was more similar to control mice on CD than control mice on HFD (Fig. 3E).

Conclusions:
Cardiomyocyte CerS5 depletion is crucial in maintaining cardiac and systemic metabolic homeostasis in mice with cardiometabolic pathophysiology. Excessive autophagy is a novel mechanism through which this may occur.
Abstract Tu108: Improved Skeletal Muscle Metabolism and Exercise Capacity Following Hydrogen Sulfide Therapy in Cardiometabolic HFpEF
Authors: Allerton Timothy , Quiriarte Heather , Li Zhen , Stampley James , Davis Gregory , Noland Robert , Doiron Jake , Sharp Thomas , Xia Huijing , Shah Sanjiv , Irving Brian , Lefer David
Published: October 2024

Introduction:
Exercise intolerance is the cardinal feature of cardiometabolic HFpEF that is largely driven by impairments in skeletal muscle metabolism and function. Hydrogen sulfide (H
2
S) is an essential regulator of mitochondrial function and metabolic homeostasis. We have previously demonstrated reductions in skeletal muscle enzymatic H
2
S production and H
2
S bioavailability in rodent models of HFpEF. The potential for H
2
S donor therapy to restore skeletal muscle metabolism and improve exercise capacity in HFpEF has not been explored.

Hypothesis:
We tested the hypothesis that oral H
2
S donor (SG-1002) therapy would restore critical aspects of skeletal muscle metabolism and exercise performance in cardiometabolic HFpEF.

Methods:
Five weeks after the onset of HFpEF (high-fat diet + L-NAME) male C57BL6/J mice (n=12 per group) were administered dietary SG-1002 (95 mg/kg/day) for 5 weeks and compared to HFD + L-NAME alone mice (i.e., control). Exercise performance was assessed using forced treadmill and voluntary wheel running (VWR) with continuous monitoring of indirect calorimetry. Skeletal muscle (soleus and gastroc) mitochondrial function was assessed by high-resolution respirometry. Palmitate (fatty acid oxidation, FAO) and leucine (BCAA) oxidation were determined by radiolabeled isotope (
14
C) assays. A force grid meter was used to determine muscle grip force production. GC-chemiluminescence was used to measure H
2
S bioavailability.

Results:
SG-1002 therapy significantly (p < 0.001) increased circulating H
2
S bioavailability (0.57 ± 0.15 vs. 0.22 ± 0.08 µM) when compared to HFpEF control mice. Forced treadmill running (125 ± 87 vs 64 ± 33 meters), grip force production (9.6± 1.1 vs. 7.5 ± 1.3 g/g lean mass) were increased (p < 0.05 vs. control for both) following SG-1002 treatment. During VWR, whole-body fat oxidation was increased (p=0.0008, 0.3 ± .01 lower RER) in SG-1002-treated mice. At the skeletal muscle level, SG-1002 treatment reduced LEAK state respiration by 52% (p < 0.0001 vs. control) and increased complete FAO by 30% (p < 0.05) while reducing incomplete FAO by 33% (p < 0.05 between groups).

Conclusion:
We demonstrate that H
2
S donor therapy significantly improves exercise capacity and underlying skeletal muscle dysfunction in a mouse “two-hit” model of cardiometabolic HFpEF.
Abstract Tu111: Effect Of Grandmaternal Diabetes and High Fat Diet on Cardiometabolic Dysfunction in the Second Generation (F2) Newborns
Authors: Ayyappan Prathapan , Larsen Tricia , Klein Abigail , Gandy Ty , Baack Michelle
Published: October 2024

Infants exposed to diabetic pregnancy are at higher risk of cardiomyopathy at birth and early onset cardiovascular disease (CVD) as adults. We have previously used a rat model to demonstrate that a combination of high fat diet (HFD) and late gestational diabetes causes impaired cardiac function in first generation (F1) newborn and adult offspring via mitochondrial dysfunction and metabolic stress. The objective of this study was to determine whether grandmaternal HFD and diabetes can cause inheritable CVD in second generation offspring (F2), even if the first generation is cross fostered during lactation and fed a standard control diet throughout life. Myocardial function in the F2 newborns was assessed using echocardiography and mitochondrial function in the isolated neonatal cardiomyocytes (NRCM) was assessed using extracellular flux analyses. Exposed F2 newborns had a higher birthweight (p=0.03) and blood glucose (p<0.0001) compared to control newborns and F1 generation parents (p=0.0002 and p<0.0001). Of significant interest, exposed F2 newborns had higher heart rates (p<0.0001), heart:body weight ratios (p<0.0001) and systolic dysfunction which was evident from lower ejection fraction (p<0.0001) and fractional shortening (p<0.0001) compared to control F2 newborns. While control and combination exposed NRCM had similar basal respiration, exposed F2 NRCM had a higher proton leak (p=0.0004) which indicates damage to the inner mitochondrial membrane and/or electron transport chain complex. There was also shift to higher glycolysis at baseline (p=0.005), but blunted glucose-induced glycolytic rate (p=0.04) in F2 newborns compared to controls. Overall results suggest that maternal HFD and diabetes causes multigenerational programming of cardiometabolic dysfunction in F2 offspring despite the F1 parent having normal diet, weight, and euglycemia during the pregnancy.
Abstract Tu113: Metabolomic Profile of Human End-Stage Ischemic Cardiomyopathy Reveals Few Differences from Non-Ischemic Cardiomyopathy
Authors: Tanosaki Sho , Bedi Kenneth , Margulies Kenneth , Arany Zoltan
Published: October 2024

Background:
The heart is highly metabolic, relying on a range of fuels to sustain its high generation of ATP. The primary source of energy in the human heart is fatty acids, but fatty acid oxidation is reduced in failing hearts, contributing to a decrease in ATP production. Previously, we provided a comprehensive metabolic profile of human non-ischemic cardiomyopathy (NICM) hearts, including maintained ATP levels despite decreased energy charge (EC), lower acylcarnitines, acyl CoAs, and free fatty acids, suggesting a defect in fatty acid import.

Research Questions:
The pathogenesis of ischemic cardiomyopathy (ICM) is dramatically different from that of NICM, raising the question of whether metabolic defects differ between these two conditions.

Aims:
Our current study was therefore aimed at elucidating the metabolomic profile of end-stage ICM compared to that of NICM.

Methods:
Whole human hearts were obtained either from patients with end-stage heart failure (NICM or ICM) undergoing cardiac transplantation or from brain-dead organ donors. Hearts were arrested in situ and kept in ice-cold cardioplegia solution at all times. Transmural tissue samples were obtained from left ventricular free wall, snap frozen, and extracted metabolites were quantified by liquid chromatography coupled with mass spectrometer.

Results:
The comparison of metabolomics data from non-failing (NF) control heart samples and NICM samples were consistent with our previous study, with marked decrease in acylcarnitines and increase in lysophosphatidylcholine. On the other hand, the metabolomics profiles of ICM and NICM samples were remarkably similar. Phenylacetylglutamine and trimethylamine N-oxide, both related to atherosclerotic disease risk, were statistically significantly higher in ICM samples compared to NICM samples.

Conclusion(s):
The human cardiac metabolome of end-stage ICM is nearly identical to that of NICM, despite dramatically different pathogeneses, suggesting that end-stage heart failure constitutes a final common pathway. It would be of great interest to evaluate metabolomic profiles of the diseases differ at earlier timepoints.
Abstract Tu118: The Mitochondrial LonP1 Is Indispensable For Cardiac Maturation And Function
Authors: Muthu Sakthijothi , Tran Zinnia , ODwyer Kienan , Guppi Sanketh , Pal Subhankhi , Velayutham Murugesan , Meadows Ethan , Hollander John , Sundararajan Venkatesh
Published: October 2024

Background:
LonP1 is a major mitochondrial ATP-dependent protease critical for mitochondrial protein quality control, However its role in cardiac function is unclear.

Hypothesis:
We hypothesize that LonP1 is essential for postnatal cardiac maturation and function.

Methods:
We employed cardiac-specific LonP1 knock-out (LonP1cKO) and Cre-control mice, assessing their heart histology (HE, trichrome), cardiac function via echocardiography, mitochondrial function by measuring oxygen consumption rate (OCR), mitochondrial complexes (I-V) activity by (ETC complex maximal activity), Iron-Sulfur (Fe-S) clusters and free radical levels by electron paramagnetic resonance (EPR) spectroscopy. The significant difference between the groups was established at p<0.05 using the Student's 't' test.

Results:
We found that LonP1cKO mice were not embryonically lethal; however, they exhibited an early mortality rate, died at an average of 21 days after birth with significantly lower body weight (7.02±0.16 gm vs. control (11.41±0.25 gm), p<0.0001, n=24) and increased heart weight to the tibia length ratio (0.080±0.002 gm/cm vs. control (0.047±0.0014 gm/cm), p<0.001, n=24). Echocardiography analysis showed that LonP1ckO mice had severely decreased left ventricular ejection fraction (%EF) (14.06±1.82% vs. control (70±2.9%), p<0.0001, n=16) and fractional shortening (% FS) (6.05±0.8% vs. control (39.15±2.5%), p<0.0001, n=16). Histological analysis revealed severe ventricular dilation and increased cardiac fibrosis. Transmission electron microscope analysis showed a significant loss of cristae and accumulation of electron-dense aggregation within mitochondria of LonP1cKO but not in control mice. In addition, LonP1cKO hearts showed reduced Fe-S clusters level (10.70±0.85 a.u vs. 21.27±1.91, p<0.0001, n=6), complex I-V activities (p<0.001), and electron flow (152.2±4.04 nm/min/mg vs. 265.3±23.8, p<0.001, n=6) compared to control.

Conclusion:
Our findings indicate that the deletion of LonP1 makes the heart unable to transition from glycolysis to substrate oxidation during the early postnatal maturation process, leading to dilated cardiomyopathy and death. This highlights the pivotal role of LonP1 in both cardiac maturation and function.
Abstract Tu124: The Transcriptional Regulator FOG2S Represses GATA4-ERRα-PGC1α Complex to Decrease Mitochondrial Metabolism
Authors: Gurunathan Sharavana , Sakamoto Tomoya , Conlon Donna , Kelly Daniel , Guerraty Marie
Published: October 2024

Background:
Cardiac transcription factors (CTFs) and coregulators drive the developmental transition from immature to mature cardiomyocytes (CMs). The nuclear receptor estrogen-related receptor (ERR), with coregulator PGC-1α and CTF GATA4, has been shown to serve as a critical role in CM maturation. Friend of GATA 2 (FOG2), a transcriptional coregulator, is crucial for normal cardiac development and exists in 2 isoforms: FOG2 and FOG2S, a shortened isoform lacking the N-terminal repressor domain and more prevalent in developing heart. Little is known about the role of FOG2S. We sought to investigate the mechanisms whereby FOG2S serves as a transcriptional regulator and assess its potential interaction with maturation CTFs.

Methods:
FOG2S was overexpressed in 8-week-old male C57/BL6 mice and human iPSC-derived cardiomyocytes (iPSC-CMs) using cardiac-specific AAV (cTNT.AAV9). Hearts were processed for RNA-seq and electron microscopy. iPSC-CMs were also treated with 0.1 mM DMOG for 16 h to induce hypoxia, essential for metabolic flexibility, and assessed with Seahorse Bioanalyzer. Hearts and iPSC-CMs were also analyzed for oxidative phosphorylation genes by qRTPCR. Co-transfection studies using a 3XERR-RE upstream of a minimal promoter driving luciferase reporter was performed in HEK293 cells with 3xFLAG-ERRα, PGC-1α-Myc/His-, GATA4-HA-, FOG2-FLAG- and/or FOG2S-FLAG-tagged plasmids.

Results:
FOG2S mice showed 678 differentially expressed genes (p<0.001, 424 genes up and 254 genes down). Gene set enrichment analysis identified oxidative phosphorylation as the top hallmark pathway, driven by decreased Nduf’s and Cox’s and increased Bnip3, a mitophagy gene. FOG2S hearts had smaller and rounder mitochondria with less cristae.
In vitro
, DMOG-treated FOG2S iPSC-CMs exhibited decreased basal and maximal respiration and reduction in mitochondrial network. There was a significant 40-80% decrease in mitochondrial gene expression, MFN1, COX10, TIMM21, TOMM22, NDUFA11, NDUFAB1. GSEA for transcription factor targets identified ERRα as the top hit. Luciferase reporter assay for 3XERR-RE activity showed FOG2S inhibited ERRα-PGC1α-GATA4 mediated activation in a dose-dependent manner. Inhibition was also mediated through GATA4 and independent of PGC1α.

Conclusion:
FOG2S overexpression suppresses oxidative phosphorylation and mitochondrial metabolism by inhibiting GATA4 in the context of ERRα-PGC1α-GATA4 complex. These results suggest that FOG2S modulates CM metabolic maturation.
Abstract Tu132: Estrogen modulation of ethanol-evoked cardiac oxidative stress and dysfunction: Role of circadian clock period-2 and ferroptosis in estrogen deficient rats
Authors: Ahmed Syed Anees , Abdel-Rahman Abdel
Published: October 2024

Background:
Alcohol triggers exacerbated cardiovascular effects in females, compared to males, in estrogen (E2) dependent manner. E2 provides cardiac protection in premenopausal women and models of menopause, yet the interplay between the circadian clock proteins and E2 in the female heart, particularly in the presence of ethanol (EtOH) remains unclear.

Aim:
This study aims to determine if the suppression of the circadian clock protein Period 2 (Per2) and associated redox enzymes contribute to E2-mediated exacerbation of the EtOH-induced cardiac ferroptosis, and dysfunction.

Methods:
Two groups of female Sprague-Dawley rats (n=6-8) were subjected to bilateral ovariectomy (OVX) and fed with EtOH (5% in liquid diet) in the presence of E2 or its vehicle for 8 weeks. Measurements of cardiac function using radiotelemetry and echocardiography along with ex vivo biochemical/molecular studies.

Results:
Compared to vehicle treated OVX+EtOH group, the E2-treated OVX+EtOH group exhibited: (1) reduced (p<0.05) body weight gain and fat mass; (2) higher (p<0.05) telemetry-measured blood pressure and heart rate; (3) reductions (p<0.05) in echocardiography derived contractility indices, ejection fraction and fractional shortening. In the same group, ex vivo molecular analysis demonstrated: (1) lower cardiac Per2 and suppressed (p<0.05) redox enzymes; (2) increased (p<0.05) ROS level and glutathione depletion (p<0.05), leading to the degradation of glutathione peroxidase 4 (GPX4); (3) heme oxygenase-1 upregulation leading to iron overload (p<0.05). These ferroptosis-related effects resulted in increased lipid peroxidation (p<0.05) compared to findings in OVX+EtOH rats in the absence of E2.

Conclusion:
This study is the first to suggest that abrogation of the E2-mediated Per2 upregulation unraveled cardiac ferroptosis and contributes to the detrimental cardiac effects of ethanol in E2-replete rats. These novel findings underscore the potential risk for myocardial ferroptosis and dysfunction in menopausal women who are consuming ethanol while on E2 replacement therapy.
Abstract Tu133: Cardiac Adaptations and Mitochondrial Protection Through Long Noncoding RNAs Regulation in Mice on a Ketogenic Diet
Authors: Tran Talan , Noto Natalia , Hernandez Ariana , Pulido-Correa Victoria , Vogel Chana , Lawrence Zachary , James Shalet , Uribe Iliana , Speth Robert , Robison Lisa , Gurusamy Narasimman
Published: October 2024

Background:
Heart failure, characterized by a metabolic imbalance, leads to impaired mitochondrial ATP production. Ketone bodies not only serve as an alternate energy source but also mitigate heart failure by reducing cardiac remodeling and inflammation. The role of long noncoding RNAs (lncRNAs) in cardiac remodeling and mitochondrial metabolism is increasingly appreciated, and this study examines the effect of ketogenic diet on cardiac lncRNA regulation and mitochondrial protection.

Methods and Results:
In a six-month study, 10-week-old male and female C57BL/6J mice were assigned to either a control diet (10% fat, 20% protein, 70% carbohydrate) or a ketogenic diet (Keto; 80% fat, 15% protein, 5% carbohydrate). Data were analyzed using unpaired two-tailed t-tests (GraphPad Prism) and reported as mean ± SE. The keto diet significantly increased blood ketone bodies (n=36/group), indicating ketosis, and led to weight gain as well as a significant decrease in heart mass relative to body weight, suggesting cardiac metabolic adaptation
(Fig 1A-B)
. The cardiac expression analysis of lncRNAs Anril, Caren, Carmn, Gm15441, Malat1, Miat, and Oip5-as1 via qPCR (n=13/group) revealed significant upregulation of Anril and Malat1 in the Keto hearts (
Fig 1C-D
), indicating their potential regulatory roles under ketogenic conditions. Sex-specific differences were not observed in the lncRNA expressions. Key proteins involved in mitochondrial biogenesis, protection, and metabolic adaptation, including Nrf2 and PGC1α, were significantly increased at both mRNA and protein levels (n=6-10) in the hearts of mice on keto diet (
Fig 1E-H
).

Conclusion:
A six-month ketogenic diet in mice promotes cardiometabolic adaptations characterized by a reduction in relative heart mass and activation of key proteins involved in mitochondrial biogenesis and protection possibly through lncRNAs Anril and Malat1. These findings shed light on novel molecular targets and the potential of ketogenic diet/ketone bodies in treating heart failure.
Abstract Tu145: Exploring Naltrindole’s hypercontracture attenuating effects during myocardial ischemia via a novel mechanism that produces cardioprotective effects in myocardial ischemia/reperfusion injury
Authors: Stinson Cameron , Le Mai An , Clair Logan , Ramsarran James , Boakye Tanoh Desmond , Melnik Juliet , Dean Tameka , CHEN Qian , Barsotti Robert , Young Lindon
Published: October 2024

Introduction:
Previously, naltrindole (NTI) produced robust cardioprotective effects (e.g.~80% reduction of infarct size) in both
ex-vivo
(2.5-5µM) and
in-vivo
(3.75-7.5 mg/kg) rat myocardial ischemia-reperfusion (MIR) injury models. Additionally, we recently showed that NTI 100-200µM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors.

Hypothesis:
We hypothesize NTI’s novel MOA is a reduction of intracellular calcium (Ca
2+
). Testing this hypothesis, we conducted isolated perfused MIR assays using KB-R7943(KB). KB is known to reduce intracellular Ca
2+
via inhibition of reverse mode Na
+
/Ca
2+
exchanger and, therefore, should attenuate myocardial ischemic hypercontracture, infarct size, and left ventricular end diastolic pressure (LVEDP). We predict that both NTI and KB will improve LVEDP and decrease infarct size compared to vehicle (dH
2
O) control and naloxone (NX) used as a negative control.

Methods:
Hearts were isolated from anesthetized male Sprague Dawley (SD) rats (~300g). We then infused NTI 1.25-5µM, KB 10-20µM, or NX 10µM for 5 min into perfused hearts, just prior to global I(30min)/R(45min). Cardiac left ventricular function was measured via a pressure transducer and infarct size using 1% triphenyltetrazolium chloride staining in the MIR assay. Data were analyzed using ANOVA and Fishers post-hoc analysis, with p<0.05 considered statistically significant.

Results:
MIR assay: NTI 5µM, 2.5µM, 1.25µM and KB 20 µM reduced infarct size (4.8±3%, n=5, p<0.05), (6.8±0.8%, n=6, p<0.05), (12.2±3%, n=5), and (2.9±2%, n=5, p<0.05) respectively compared to vehicle (14.1±2%, n=9) and NX 10µM controls (18±3%, n=3). NTI 5 µM and KB 20 µM had significantly improved LVEDP to (32.4±11%, n=5, p<0.05) and (22.8±4%, n=5, p<0.05) respectively compared to vehicle control (72.4±6%, n=9) and NX 10µM controls (54.2±8%, n=3)

Conclusion:
Both NTI (2.5µM, 5µM) and KB (20µM) reduced infarct size and improved LVEDP. The novel MOA of NTI may be mediated via a reduction in intracellular Ca
2+
during myocardial ischemia. The infarct reducing effect of NTI can benefit patients undergoing elective PCI, CABG, and transplant procedures.
Abstract Tu148: cGAS-STING pathway promotes granulopoiesis and neutrophil differentiation in acute ischemia
Authors: Zhu Jiankun , Parra pablo , Luo Xiang , Cao Dian
Published: October 2024

Contemporary evidence supports neutrophils' complex roles beyond being the simple and powerful destruction tools in MI. However, our understanding remains limited. We especially lack the knowledge of how cGAS-STING regulates neutrophil functions in acute ischemia. The cGAS-STING pathway senses cytosolic DNA to produce IFN and ISGs and is a pillar of innate immunity. Early evidence suggested neutrophils did not express cGAS. Taking advantage of the improved detecting method, we revisited this topic because this pathway can play an essential role in neutrophil function in MI. Using purified neutrophils from WT and knockout mice (as control), we detected cGAS, STING, and IRF3 in these cells. To determine if cGAS-STING regulates neutrophil function, we first generated MI model in cGAS
-/-
, STING
-/-
mice, and their WT littermates and performed flow cytometry and RNA-seq analyses. We uncovered that inhibiting cGAS-STING decreases neutrophil production in bone marrow (BM) after ischemia. Interestingly, cGAS- and STING-deficient neutrophils express significantly higher levels of primary granule molecules such as
MPO, Prtn3, Elane, Cts- (s, g, b, l)
but much lower levels of the secondary and tertiary granule components
,
including
Mmp9, Mmp8, Olfm4, Cd177, Lyz2, and Lyz1.
Neutrophils acquire granules sequentially during differentiation. Therefore, our data suggest inhibiting cGAS-STING leads to a stagnated neutrophil differentiation in the BM and mobilizes CD101
-
immature neutrophils into the circulation and MI hearts. Consistently, cGAS- and STING-deficient neutrophils release less MMP8 and MMP9 into the ischemic area. Ischemia does not trigger the degranulation of primary granules, as indicated by the minimal changes of CD63. Our data further suggest that TF C/EBP𝜹 functions downstream of cGAS-STING to promote neutrophil differentiation. Adoptive transfer experiments suggest the neutrophil-specific function of cGAS-STING. In conclusion, we identify cGAS-STING as a novel mechanism promoting emergency granulopoiesis and neutrophil differentiation after MI and uncover its functional implications in ischemia-induced remodeling. Studies using Mrp8-Cre-mediated cGAS-STING deletion in granulocyte/macrophage progenitors are in progress.
Abstract We008: Hallmark Maturation of the Human Pluripotent Stem Cells-derived Cardiovascular Progenitors in Myocardial Infarcted Large Animal Model
Authors: Adusumalli Swarnaseetha , Lim Samantha , Ye Lei , lim Eric , Ren Shuxun , Wang Yibin , Petretto Enrico , Tryggvason Karl , Yap Lynn
Published: October 2024

For the goal of regenerative cardiology, cellular therapy may be used to restore injured heart muscle. The molecular mechanisms behind the engraftment and maturation of these transplanted cells, however, remain poorly understood. Thus, to obtain a comprehensive understanding of the complex transcriptional landscapes within their transplanted spatial context, we utilized spatial transcriptomics in this study.

We have previously demonstrated the safety and effectiveness of transplanting our cardiovascular progenitors (CVPs) produced from laminin-221 into pig hearts that had suffered myocardial infarction (MI). Here, we undertake 10x spatial transcriptomics in chronic MI pig models at 1-, 4-, and 12 weeks post-transplantation to further our understanding of these cells in vivo.

With confidence, we can distinguish between the transcripts from the pigs and the humans in the infarcted region. By using human-specific anti-Ku80 antibodies for immunohistochemical labeling of the pig tissue samples, this in silico prediction was verified. The CVPs' engraftment and longevity in the infracted region were shown by the long-term identification and staining of human cells at various time points. It is interesting to note that after 12 weeks, ribosomal and mitochondrial activity were higher than at 1 and 2 weeks. These results are consistent with the hallmarks of cardiac maturation, in which cells transit from immature fetal-like cells to mature cardiomyocytes (CMs) by switching their metabolic energy source from glucose to fatty acids. Also, by comparing the heart-specific genes, including MYH6 and MYH7, we were able to quantify the myosin isoform switches in the xenograft over time. Furthermore, to delineate the fate of the transplanted cells, we performed a ligand-receptor interaction analysis. Intriguing, we identified the midkine protein as a possible secreted human paracrine factor that enhances angiogenesis in the graft.

In conclusion, we have shown the successful engraftment and maturation of human CVPs toward CMs using spatial transcriptomic technology. These data will be used to understand other aspects of regenerative cardiology such as the mechanism of VTs and in vivo cellular repair mechanisms. A shiny app atlas has been generated to share our transcriptomics data for all to explore gene expression of human transplanted cells in myocardial infarcted pig hearts.
Abstract We010: In Vivo Partial Reprogramming of Cardiomyocytes to a Molecularly Rejuvenated State Ameliorates Cardiac Failure
Authors: De Lazaro Irene , Zhang Bohan , Makarova Nadezhda , Mariotti Marco , Gladyshev Vadim , Mooney David
Published: October 2024

Introduction:
i
n vivo
partial cell reprogramming through transient overexpression of
Oct3/4
,
Klf4
,
Sox2
and
cMyc
(OKSM) transcription factors generates de-differentiated or molecularly rejuvenated cells that may contribute to tissue regeneration. However, not all cell types are equally amenable to reprogramming, and tools to induce cell type-specific reprogramming
in vivo
are scarce. Here, we generated cardiomyocyte-specific, “reprogrammable” mice (CM-OKSM) that enabled inducible OKSM expression specifically in this cell type, characterized by its remarkably poor regenerative capacity after birth. Using this model, we investigated if partial,
in situ
, cardiomyocyte reprogramming was feasible and able to enhance cardiac regeneration.

Methods:
To generate lineage-traceable CM-OKSM mice, we combined Cre recombination driven by a cardiomyocyte-specific promoter (
Myh6
), conditional GFP labelling and conditional, doxycycline (dox)-inducible, control of OKSM expression. We administered 1 mg/ml dox in the drinking water of CM-OKSM mice, and investigated reprogramming at the molecular level (immunostaining, RT-qPCR, RNAseq, DNA methylation), histological level (H&E, electron microscopy) and functional level (teratoma assay, echocardiography). We compared dox administration for six cycles of a two-day ON/five-day OFF regime, and for eighteen continuous days, to investigate partial and full reprogramming to pluripotency, respectively. We conducted studies in young adult mice (8 to 12 weeks old) and on older mice (9 months old) with heart failure, to assess the effects of cardiomyocyte reprogramming in cardiac performance.

Results and Discussion:
In young CM-OKSM mice, adult cardiomyocytes can be fully reprogrammed to pluripotency
in vivo
by eighteen-day overexpression of OKSM. This was evidenced by significant upregulation of endogenous pluripotency markers, loss of sarcomere organization, cell de-differentiation, and the appearance of GFP+ teratomas with trilineage contribution. We then found that cyclic OKSM induction prevented reacquisition of pluripotency and tumorigenesis, both in young and old mice. In the latter, partial reprogramming of cardiomyocytes induced significant rejuvenation of their epigenetic age and halted the progression of congestive heart failure. Our results confirm that the OKSM cocktail can reprogram adult mouse cardiomyocytes
in vivo
and that partial reprogramming can contribute to functional improvements in cardiac disease.
Abstract We022: Depletion of Tip60 After Myocardial Infarction Induces Histone H2A.Z Deacetylation Followed by Cardiomyocyte Dedifferentiation/Cell-Cycle Activation
Authors: Wang Xinrui , Kulik Katherine , Wan Tina , Lough John , Auchampach John
Published: October 2024

Myocardial infarction (MI) leads to cardiomyocyte (CM) loss, resulting in cardiac dysfunction and heart failure. Remuscularization of injured myocardium requires proliferation of surviving CMs. However, approaches aimed at inducing CM regeneration following MI have been inadequate. To this end, we are targeting the acetyltransferase Tip60 (Tat-interactive protein 60 kD), a pleiotropic tumor suppressor encoded by the
Kat5
gene. Using a murine genetic model, we previously reported that disruption of
Kat5
promotes CM cell-cycle re-entry, and protects against the damaging effects of MI. It is becoming increasingly recognized that pre-existing CMs must undergo dedifferentiation accompanied by reversion to glycolytic metabolism in order to resume proliferation. The site-specific acetylation of the histone variant H2A.Z at lysine K4/K7 (H2A.Zac
K4/K7
) has been shown to maintain the differentiated state in hematopoietic stem cells (HSCs), neurons, and skeletal myocytes. Here, we report that Tip60 depletion post-MI results in the near-complete absence of H2A.Zac
K4/K7
in CMs. This is associated with the enrichment of differentially expressed genes (DEGs) in epithelial to mesenchymal transitioning (EMT), cytoskeletal disassembly, extracellular matrix (ECM) softening, and metabolic reprogramming. To assess therapeutic relevance, we have begun to evaluate the potential cardioprotective effects of the anti-parasitic drug pentamidine, a known Tip60 inhibitor. Systemic administration of pentamidine on days 3-16 post-MI preserved cardiac function, accompanied by CM cell-cycle activation. These data suggest that pentamidine treatment enhances remuscularization, thereby promoting the retention of post-MI function, supporting the translational promise of targeting Tip60 as an innovative therapy for ischemic heart disease.
Abstract We023: P53 Activation Promotes Maturational Characteristics of Pluripotent Stem Cell-derived Cardiomyocytes in 3D Suspension Culture via FOXO-FOXM1 Regulation
Authors: Velayutham Nivedhitha , Garbern Jessica , Elwell Hannah , Zhuo Zhu , Ruland Laura , Elcure Alvarez Farid , Frontini Sara , Rodriguez Carreras Yago , Ricci-Blair Elisabeth , Shaw Jeanna , BOUFFARD Aldric , Morgan Sokol , Mancheno Juncosa Estela , Rhoades Seth , van den Berg Daphne , Kreymerman Alexander , Aoyama Junya , Hoefflin Jens , Ryan Herb , Ho Sui Shannan , Lee Richard
Published: October 2024

Background:
Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)
in vitro
that recapitulate key characteristics of mature
in vivo
cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation.

Hypothesis:
The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture.

Methodology:
3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively.

Results:
P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups.

Conclusions:
These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.
Abstract We030: Microtubule Organizing Centers in Cardiomyocyte Proliferation and Maturation
Authors: Li Siqi , Avila Enrique , Berthiaume Anna , Albrecht Katelyn , Liu Chun , Han Lu
Published: October 2024

Mammalian cardiomyocytes undergo a loss of proliferative capacity due to cell division failure, leading to polyploidy (genome duplication) during postnatal maturation. Centrosomes, primary microtubule organizing centers (MTOCs) in animal cells comprised of centrioles and pericentriolar matrix (PCM), play a crucial role in cell division. In differentiated cells, non-centrosomal (nc) MTOCs can reorganize microtubules, with cellular localization varying between cell types to mediate specialized functions. The specific role of ncMTOCs in cardiomyocyte division and maturation remains unknown.

This study investigates the formation and function of ncMTOCs in cardiomyocytes, aiming to elucidate the regulating mechanism. Our results demonstrate that during the maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), centrosomes disassemble, and their PCM translocates to the nuclear envelope, establishing perinuclear ncMTOCs. Furthermore, maturing CMs with ncMTOCs often exhibit polyploidy, indicating incomplete cell division. Microtubule dynamic experiments reveal that ncMTOCs serve as organizing centers for microtubules, promoting their assembly around the nucleus in maturing cardiomyocytes.

To understand the mechanism regulating the transition from centrosomes to ncMTOCs in cardiomyocytes, we identified a significant downregulation of the nuclear lamina protein Lamin B2 (LMNB2) during CM maturation. Our results indicate that LMNB2 is essential for mitotic spindle formation in CMs but not in stem cells, suggesting a distinct role for LMNB2 in CMs. Our results also suggest an increased centrosome to ncMTOC transition in LMNB2-depleted CMs at an early differentiation stage.

Ongoing research aims to investigate the impact of perinuclear ncMTOCs on cardiomyocyte proliferation through pericentriolar matrix knockdown in hiPSC-CMs. Additionally, the study seeks to further elucidate the role of Lamin B2 in the transition of centrosome proteins and its underlying mechanism.
Abstract We032: FOXK1-Gli2 network regulates cardiomyocyte proliferation and heart regeneration
Authors: Larson Thijs , Das Satyabrata , Sierra-Pagan Javier , Choi Young Geun , Ma Xiao , Bieda Alice , Alexy Tamas , li qinglu , Sadek Hesham , Zhang Jianyi , Garry Mary , Garry Daniel
Published: October 2024

Unlike adult mammals, the neonatal mouse has an exceptional regenerative capacity following myocardial injury. Our goal was to decipher and define novel factors and mechanisms that have a role in regeneration of the injured adult mouse heart. FOXK1 is a transcription factor that regulates cell cycle kinetics, myogenic stem cell proliferation, and skeletal muscle regeneration. During development, its expression is restricted to progenitors, somites and the heart. In these studies, we identified a novel role for FOXK1 as a regulator of cardiomyocyte (CM) proliferation and regeneration through the regulation of
Gli2
. Analysis of single nucleus RNAseq datasets of neonatal hearts revealed that FOXK1 was expressed in neonatal CMs. qPCR analysis from P1 hearts of control and
Foxk1
null mice confirmed this finding and demonstrated dysregulation of the cell cycle regulators,
Ccne1
and
p21
, in the absence of
Foxk1
. Control and
Foxk1
null mice were pulsed with EdU from P3 to P7 which demonstrated that the absence of
Foxk1
led to a decrease in CM proliferation in-vivo. Then, using a transgenic mouse model that we engineered to overexpress FOXK1 in muscle (
MCK-Foxk1
), we observed CM proliferation in adult
MCK-Foxk1
mice, which showed increased incorporation of EdU and increased pH3 expression compared to control. Bulk RNA-seq analysis of P1, P7, and P14 hearts indicated increased CM proliferation in the
MCK-Foxk1
mouse and revealed
Gli2
as a potential downstream target of FOXK1. A gel shift assay was performed, which demonstrated that FOXK1 was able to bind to a FOXK1 motif upstream of the
Gli2
gene. ChIP-PCR further verified that
Gli2
was a direct downstream target of FOXK1 in the embryonic stem cell-embryoid body (ES-EB) system. We then performed ligation of the left anterior descending coronary artery (LAD) in adult control and
MCK-Foxk1
mice. Echocardiography following the LAD-ligation injuries showed improved cardiac function in
MCK-Foxk1
transgenic mice compared with controls. These results identify FOXK1 as a regulator of CM proliferation and heart regeneration and suggest that this function is mediated through the regulation of
Gli2
gene expression.