Search Results for: Come stress e attacchi di cuore sono collegati

Circulation, Volume 150, Issue Suppl_1, Page A4144377-A4144377, November 12, 2024. Mammalian cells have several adaptive mechanisms to counteract the adverse effects of metabolic and redox stress induced by ischemia. Hypoxia, a hallmark of ischemia, results in the selective reduction of the tricarboxylic acid cycle metabolite, α-ketoglutarate, to theL-(S)-enantiomer of 2-hydroxyglutarate (L2HG) in several cell types. L2HG protects hypoxic cells by buffering increases in the NADH/NAD+redox couple and inhibiting mitochondrial electron transport. In addition, the accumulation of L2HG induced by genetic knockout of L2HG dehydrogenase (L2HGDH), the only known enzyme capable of oxidizing L2HG back to α-ketoglutarate, protects mice against myocardial injury during ischemia. This protection by L2HG manifests as decreased myocardial infarct size and improved cardiac function, owing partly to a metabolic shift in carbon flux from glycolysis towards the pentose phosphate pathway. However, myocardial ischemia also leads to perturbations in fatty acid metabolism as manifest by accumulation of acylcarnitines and acyl-CoA’s. It remains unclear as to whether or not L2HG accumulation affects fatty acid metabolic homeostasis during myocardial ischemia. Here, we induced L2HG accumulation by homozygous deletion of thel2hgdhgene in male mice (l2hgdh-/-; n=12). Hearts isolated from these mice and their wild-type littermates (l2hgdh+/+; n=13) were subjected toex vivoLangendorff perfusion at coronary perfusion pressure of 80 mmHg for 30 min with (ischemic group) or without (control group) subsequent perfusion at ~10% pressure for 90 min. Using liquid chromatography tandem mass spectrometry (LC-MS/MS)-based nontargeted lipidomics, we identified several species of long chain acylcarnitines that accumulated by ischemia in hearts obtained fromL2HGDH+/+mice [linoleoyl carnitine, palmitoyl carnitine, and hydroxy-linoleoyl carnitine increased by 13.2-fold (p=4.2 x 10-6), 10.7-fold (p=1.7 x 10-6), and 10.0-fold (p=5.8 x 10-6), respectively]. Interestingly, however, this accumulation was attenuated substantially in ischemic hearts obtained fromL2HGDH-/-mice [hydroxy-linoleoyl carnitine, palmitoyl carnitine, and linoleoyl carnitine were downregulated by 62.6% (p=0.034), 54.2% (p=0.018), and 35.4% (p=0.054) in ischemic hearts fromL2HGDH-/-mice when compared toL2HGD+/+littermates]. Overall, these findings highlight a novel and potentially important role for L2HG in restoring the perturbations in fatty acid metabolism induced by myocardial ischemia.

Circulation, Volume 150, Issue Suppl_1, Page A4145480-A4145480, November 12, 2024. Introduction:Acute respiratory distress in the peripartum period can occur from a variety of reasons including pulmonary or cardiac causes. Careful integration of the history, physical examination and imaging studies is crucial to make a definitive diagnosis.Case presentation:The patient is a 22-yr-old G1P0 African American woman with no significant medical history who had two unsuccessful external cephalic versions and required Cesarean section. Intraoperatively, she became dyspneic and hypoxic. She had labored breathing, tachycardia, scattered wheezes and hypotension. She had acute pulmonary edema, requiring endotracheal intubation. PA catheter placement revealed cardiac output of 3.7 L/min, Pulmonary artey pressure was 37/27 mmHg and SPO2 of 80% on 100% FiO2. Transthoracic echocardiogram (TTE) revealed moderately reduced left ventricular ejection fraction, (LVEF) 30-35% and moderate mitral regurgitation. She had preserved apical LV function with reduced contractility in the basal segments suggestive of reverse Takotsubo syndrome. RV size and function were normal, making significant pulmonary embolism less likely. CXR revealed interstitial edema. EKG had sinus tachycardia. Peak troponin I was 3.3ng/mL and Lactic acid 4.2 mmol/L. Patient received IV diuretic and inotropic support with Norepinephrine, Epinephrine and Milrinone. She was extubated after 2 days. TTE four days later revealed recovery of LVEF to 53%. Three weeks later, she was asymptomatic. Cardiac MRI showed normal LVEF 58% and no wall motion abnormalities.Discussion:The patient had acute pulmonary edema and systolic dysfunction.In the absence of chest pain or ST elevation on EKG, spontaneous coronary dissection was unlikely. Takotsubo cardiomyopathy (TMC) is an acute but transient cardiac dysfunction following significant physical or emotional stress. Attempted external cephalic version may have triggered TMC. It is important to distinguish between TCM and Peripartum cardiomyopathy (PPCM) in these patients as it may prevent the continuation of long term medical therapy for heart failure. PPCM has a more gradual onset. Quick normalization of her LV function strongly favors that she experienced Takotsubo, not Peripartum cardiomyopathy.Conclusion:Making an accurate diagnosis of Takotsubo cardiomyopathy by serial echocardiography to demonstrate quick improvement in LV function has long-term implications regarding future pregnancies and prognosis.

Circulation, Volume 150, Issue Suppl_1, Page A4140616-A4140616, November 12, 2024. Introduction:Cardiovascular events occur in patients without obstructive coronary artery disease (oCAD), indicating a need for better risk stratification tools. CT angiography (CTA)-based high-risk plaque (HRP) characteristics predict such events effectively but are not widely available. We performed comprehensive proteomics profiling in the PROMISE trial to identify biomarkers associated with HRP, oCAD, and major adverse cardiovascular events (MACE).Methods:The PROMISE trial randomized patients with stable chest pain to CTA vs. stress testing. Proteomic profiling (Olink, 524 proteins) was performed on 1724 participants with CTA and biospecimens. A high-risk composite phenotype (HRCP) was defined as presence of oCAD (≥50% stenosis in any major vessel), Leaman score >5, coronary artery calcium score ≥400, or HRP features on CTA. Individual proteins were tested for association with HRCP (false discovery rate-adjusted p-value [q]

Circulation, Volume 150, Issue Suppl_1, Page A4119210-A4119210, November 12, 2024. Introduction:Insulin growth factor (IGF) binding protein-1 and 2 (IGFBP-1 and 2) are implicated in acute myocardial infarction (AMI) and have been suggested as predictors of mortality and development of heart failure. The IGF axis has been shown to play roles in cell proliferation and apoptosis. We analysed the serum IGFBP-1 and 2 levels in AMI before and after percutaneous coronary intervention (PCI) and correlated this with cardiac magnetic resonance (CMR) imaging to better establish their significance as biomarkers in AMI.Methods:Patients with AMI who presented to Royal Free Hospital, London, were recruited for proteomic analysis (O link Proximity Extension Immunoassay) at presentation, 24h and 72h following primary PCI. All underwent both admission and follow-up CMR at 6 months to assess for myocardial oedema, left ventricular volumes and function, and assessment of scar.Results:Proteomic evaluation in 20 AMI patients (male 95%; median age 59) revealed significant reduction in the average IGFBP-1 values at discharge compared with presentation, from 5.93 to 4.52 (24% reduction, p=0.003). Conversely, the average IGFBP-2 values at discharge significantly increased from 7.66 to 8.29 (8% increase, p=0.001). Higher discharge IGFBP-2 value was also correlated with increased indexed left ventricle end diastolic volume (LVEDV) at 6-month follow-up (R=0.54, p=0.032).Discussion:IGFBP-1 and 2 showed significant change following intervention for AMI. IGFBP-1 is a stress hormone, which is directly and indirectly activated by cytokines, dysglycaemia and vasopressin activation. Its subsequent reduction following PCI in AMI indicates relative resolution of inflammation, but this was not correlated with changes in infarct size or LV function at follow-up. IGFBP-2 showed significant upregulation, and higher values at discharge were significantly correlated with increased LDEDV at follow-up, suggesting adverse clinical outcomes within this setting. This data suggests differential roles of IGFBP-1 and 2 in the pathogenesis of AMI.

Circulation, Volume 150, Issue Suppl_1, Page A4135229-A4135229, November 12, 2024. Background:Endothelial glycocalyx (eGC) is a layer of glycosaminoglycans adhered to proteoglycans that cover the inner surface of the vessels. The integrity of this structure sustains some vascular properties such as shear stress-induced nitric oxide release and the prevention of platelets and neutrophils adhesion on the vessel surface. eGC shedding could contribute to atherosclerotic plaque vulnerability and acute myocardial infarction (AMI) installation. This research investigated whether acute eGC damage and microvascular dysfunction occurred during AMI.Methods:Sublingual microcirculation was assessed through Capiscope HCVS Handheld Video Capillaroscopy System (KK Technology, Honiton, UK) during the first 72h of hospital admission because of ST-elevation AMI (before) and repeated around six months later (after). All images were automatically analyzed using the GlycoCheck (Alpine, UT, USA), which estimated the perfused vascular density (PVD), the red blood cell (RBC) filling of the vessels, the perfused boundary region (PBR) in µm, an inverse parameter of the eGC thickness; and the microvascular health score (MVHS). This score ranges from 0-10; the lower its numerical value, the greater the microvascular impairment.Results:Twenty patients were included (61±10 years old, 85% males, 55% anterior AMI, 70% hypertension, 25% diabetes, 45% current smoker, all underwent primary PCI). The time between the two sublingual assessments was 188±31 days. The PBR [1.96±0.22µm vs. 1.84±0.19µm]; p=0.047 and flow-adjusted PBR [1.40±0.25µm vs. 1.24±0.16µm]; p=0.045 decreased statistically significant over time. The RBC filling [71.56±4.33% vs. 74.02±3.88%]; p=0.016 and the MVHS [4.28 (interquartile range (IRQ) 3.38-4.90) vs. 5.04 (IQR 4.37-6.00)]; p=0.044 increased statistically significant over time. The PVD [500±86 x10-2µm/mm vs. 522±84×10-2µm/mm]; p=0.255 did not modify over time.Conclusion:This finding was compatible with eGC shedding and microvascular dysfunction during AMI and later reconstitution of this structure after six months.

Circulation, Volume 150, Issue Suppl_1, Page A4141191-A4141191, November 12, 2024. Introduction:Regular physical exercise is widely recognized for its beneficial effects on overall health. Despite emerging evidence that early-life experiences profoundly impact adult and aged health, the long-term effects of early-life exercise remain uncertain.Hypothesis:We hypothesized that early-life exercise extends healthspan and attenuates cardiovascular aging in aged mice.Methods:Male and female C57BL/6J mice were subjected to a 3-month swimming exercise regimen starting from 1 month of age (1.5 hour per day, 5 days per week), while no exercise intervention was conducted at other times. The sedentary group was raised under similar conditions. The healthspan of aged male and female mice was assessed, including overall metabolic function, cardiovascular function, frailty index and other relevant measurements. Transcriptional profiling of young and old mice was performed to gain insights into the mechanisms underlying the effects of early-life exercise on aging.Results:Early-life exercise improved metabolic health in aged mice. Assessment of body composition showed increased lean mass in the exercise group. When subjected to food deprivation stress in metabolic chambers, aged mice with early-life exercise exhibited increased energy expenditure, carbohydrate oxidation, and fat oxidation levels in both males and females. Evaluation of frailty index at 24 months of age, involving 31 frailty phenotypes, indicated lower frailty index score in the early-life exercise group, suggesting an attenuation of age-related phenotypes. While no significant differences were observed in blood pressure and cardiac systolic function, early-life exercise was associated with improved cardiac diastolic function at 19 months of age. Furthermore, early-life exercise resulted in decreased heart-carotid pulse wave velocity and improved endothelial-dependent vascular relaxation in aged mice, indicating better vascular function in elderly mice with early-life exercise.Conclusions:The findings demonstrate that early-life exercise contributes to an increased healthspan, as evidenced by enhanced metabolic health and reduced frailty. Both male and female aged mice exposed to early-life exercise exhibit improved cardiovascular function.

Circulation, Volume 150, Issue Suppl_1, Page A4140682-A4140682, November 12, 2024. Introduction:Dextrocardia is a rare congenital condition where the heart’s apex points to the right, with an incidence of about 0.01%. Patients usually have a normal life expectancy unless other structural heart diseases are present. The prevalence of atherosclerotic coronary artery disease (CAD) in these individuals is believed to be similar to that of the general population.Due to its rarity, data on coronary interventions in this subset are scarce, with available literature mostly limited to case reports. Patients with dextrocardia present a diagnostic challenge, particularly in the context of acute coronary syndrome.Case Presentation:A 49-year-old male with a medical history of dextrocardia, hypothyroidism, dyslipidemia and hypertension was referred to a cardiologist by his primary physician due to a 3-week history of unstable angina. His vital signs were normal, and the physical examination was unremarkable. Electrocardiogram (ECG) showed a prominent S wave in the left-sided leads and a prominent R wave in the right-sided chest leads, suggesting dextrocardia. Although he had a normal echocardiogram and stress test a year ago at a different hospital, due to his symptoms and intermediate-high risk probability of coronary artery disease (CAD), the decision was made to proceed with a cardiac catheterization using a trans-radial approach with a horizontal sweep technique. During the procedure, a 6-French tiger catheter (TIG) was guided into the left coronary sinus and advanced into the left ventricle under fluoroscopic guidance.The left main coronary artery was widely patent bifurcating into the left anterior descending and left circumflex. The left circumflex had 80% proximal stenosis with minimal luminal irregularities in the mid to distal portion. After guidewire crossing, balloon angioplasty was performed, and a drug-eluting stent was deployed. An intravascular ultrasound was also performed, which was negative for vessel dissection. There were no post-procedure complications, and the patient was discharged on beta blockers and dual antiplatelet agents.Conclusion:Performing cardiac catheterization in patients with dextrocardia presents unique technical challenges due to the mirror-image anatomy. This case report highlights the modifications in standard techniques, emphasizing the need for specialized skills and strategies to achieve successful outcomes in such cases.

Circulation, Volume 150, Issue Suppl_1, Page A4147177-A4147177, November 12, 2024. Heart failure is a leading cause of death in the United States, and is associated with significant myocardial deterioration. cAMP and cGMP play critical roles in cardiovascular biology and disease. cAMP and cGMP form multiple spatially discrete and functionally distinct cyclic nucleotide ‘pools,’ each associated with unique multi-protein complexes comprising cyclases, PDEs, and other signaling molecules, contributing to distinct biological functions. Phosphodiesterase 10A (PDE10A) is able to hydrolyze both cAMP and cGMP. Our previous published study provides strong evidence supporting a critical role of PDE10A induction in pathological cardiac remodeling and suggest the therapeutic potential of PDE10A inhibition. However, the cellular and molecular mechanisms and the sources of cyclic nucleotides that are regulated by PDE10A remain unknown. Our encouraging results revealed that the anti-hypertrophic effect of PDE10A inhibition/deficiency is dependent on cAMP/PKA signaling in cultured adult mouse cardiomyocytes (CM). We identified the existence of adenosine-dopamine receptor (A2AR-D2R) heterodimer and the connection of PDE10A with A2AR-D2R signaling, by which PDE10A inhibition uniquely promotes heterodimerization of A2AR-D2R, and biased activation of D2R/β-arrestin2 (βarr2) through A2AR/cAMP/PKA-mediated phosphorylation of D2R in CMs. Consistent with what we found in CM, disruption of A2AR-D2R heterodimer or D2R/ βarr2-biased activation significantly abolish the protective effects of PDE10A inhibition on cardiomyopathy and dysfunctionin vivo. Additionally, we found that activation of D2R/βarr2 signaling attenuates stress-induced cardiac remodeling and dysfunctionin vivo. Together, these results strongly suggest that PDE10A plays a critical role in pathological cardiac remodeling by promoting CM hypertrophy through regulation of A2AR-D2R/βarr2 biased signaling. Targeting PDE10A and GPCR heterodimerization in CMs may represent a novel therapeutic strategy for treating cardiac diseases.

Circulation, Volume 150, Issue Suppl_1, Page A4114198-A4114198, November 12, 2024. Background:Mitochondrial dysfunction contributes to heart failure (HF) progression via diminished energy supply, release of reactive oxygen species, and increased cell death. However, there has yet to be clinically relevant biomarkers that address mitochondrial-mediated cardiac injury in patients. Humanin (HN) is a mitochondria-derived peptide that protects cells through the interference of apoptosis and reduction of oxidative stress. The objectives of our study were to measure circulating plasma HN levels in patients with HF with reduced ejection fraction and describe the relationship of HN to validated, clinically relevant measures of HF.Methods:We conducted a prospective cohort study. To be included in the study, patients must have: 1) left ventricular ejection fraction

Circulation, Volume 150, Issue Suppl_1, Page A4146892-A4146892, November 12, 2024. Background:Takotsubo cardiomyopathy (TCM) is a unique and reversible heart condition typically triggered by intense emotional or physical stress. Meanwhile, Cirrhosis, a chronic liver disease, is associated with increasing inflammation and oxidative stress, which can potentially worsen cardiovascular problems. In this study, we aimed to study the impact of cirrhosis on outcomes in TCM.Methods:A retrospective analysis was performed using the NIS database 2016 -2020 and the International Classification of Diseases, Tenth Revision codes to identify patients > 18 years old with the principal diagnosis of Takotsubo cardiomyopathy. The effect of cirrhosis was studied on in-hospital mortality and secondary outcomes such as cardiogenic shock, ventricular arrhythmia (VA), acute respiratory and acute kidney injury (AKI), as well as resource utilisation. Categorical variables were compared using the chi-square test, and continuous variables were compared using the t-test—multivariable regression analyses adjusted for demographics, hospital-level characteristics, and relevant comorbidities.Results:We identified a total of 190,025 patients with TCM. Of these, 3,335 (1.76%) patients had liver cirrhosis, with notably higher prevalence of diabetes (33.6%), CKD (20.7%), anemia (10.2%), and major depressive disorder (25.6%) in the cirrhosis group. A higher incidence of in-hospital mortality (12.9% vs. 6.3%, p

Circulation, Volume 150, Issue Suppl_1, Page A4141139-A4141139, November 12, 2024. Background:The underlying mechanisms of microvascular dysfunction in Takotsubo cardiomyopathy (TCM) remain incompletely understood.Aim:As CD34+cells are central to coronary microvascular homeostasis we investigated the potential association between CD34+cell count and changes in left ventricular function in patients with TCM.Methods:In a single-center prospective pilot cohort study we included 19 consecutive patients with TCM treated at our center between January 1st, 2022, and December 31st,2023. Patients with a history of malignancy were not considered for this analysis. TCM diagnosis was established per InterTac Registry criteria. Patients were enrolled within 24h of admission and underwent comprehensive clinical examination, blood biochemical and hematological analysis, and echocardiography at baseline and 6-month follow-up. CD34+cell count was measured using Beckman-Coulter Navios EX flow cytometry with standard antibodies according to ISAGE protocol.Results:Patient average age was 66±7 years, all patients were female, and hypertension and diabetes were present in 11 (57%) and 16 (84%) patients, respectively. The initial clinical presentation of TCM was acute chest pain in 15 (79%) patients and 16 (84%) patients displayed symptoms and signs of acute heart failure. Physical or psychological stress was identified in 17 (89%) of patients. Mean baseline left ventricular ejection fraction (LVEF) was 53±11% with apical dyskinesia being present in all patients. A peak mean troponin and NT-proBNP serum levels were 2353±2917 pg/mL and 3273±3196 pg/mL, respectively. The average baseline CD34+ cell count was 1.26±1.0×106/L. We established a significant positive correlation between baseline LVEF (r=0.54; P=0.03) and LVOT VTI (r=0.61; P=0.01) and CD34+ cell count. When stratifying patients on LVEF, patients with LVEF≥50% displayed significantly higher CD34+ cell count than patients with LVEF

Circulation, Volume 150, Issue Suppl_1, Page A4144880-A4144880, November 12, 2024. Introduction:Atherosclerosis is the leading cause of death globally. Growing evidence suggests that mitochondrial dysfunction plays a significant role in atherosclerosis. However, the progression of mitochondrial energy generation disorders during atherosclerosis development remains unclear. In 2021, our lab indicated that activation of G-Protein Coupled Receptor 55 (GPR55) in human aortic endothelial cells (HAECs) could lead to EC activation, potentially increasing inflammation by altering mitochondrial transcription. EC activation is the central pathological event in atherosclerosis. However, whether and how mitochondrial dysfunction leads to EC activationin vivoand promotes atherosclerosis through the GPR55 pathway is still unknown.Methods:Lysophosphatidylinositol (lysoPI), an endogenous ligand for GPR55 activation, was used as a stimulus (20uM for 24hrs) to treat HAECs for Seahorse mitochondrial stressin vitroanalysis. Wild-type mice, apolipoprotein E (ApoE-/-) deficiency mice, and GPR55/ApoE double knockout (DKO) mice were used for bulk RNA sequencing andEn faceanalysis.Results:DKO mice showed a significant decrease in atherogenic lesions compared to ApoE-/- mice.In vitroMito-stress indicated that GPR55 activation significantly increased basal respiration, spare capacity, maximal respiration, proton leak, and ATP production in HAECs, while inhibition of GPR55 reduced these parameters to basal levels. This suggests that GPR55 activation enhances mitochondrial function without affecting non-mitochondrial oxygen consumption, coupling efficiency, or mitochondrial membrane potential. We also generated a list of 289 genes associated with mitochondrial energy generation genetic disorders. Transcriptomic profiling of these genes in atherogenic mice fed with an HF diet for 3, 6, 12, 32, and 72 weeks showed a downward trend in significantly downregulated OXPHOS genes. This significant downregulation suggests increased OXPHOS biogenesis reprogramming as the disease progresses. Additionally, by comparing upregulated genes in ApoE-/- mice with those downregulated in DKO mice, we identified seven potential genes downstream of GPR55 signaling, including M2, MP, A1, F4, P8, H10, and S2, which are involved in the TCA cycle and metabolism, protein import, and lipid modification.Conclusion:Our study provides the first comprehensive examination of how impairments in mitochondrial OXPHOS biogenesis influence atherosclerosis progression via GPR55 signaling.

Circulation, Volume 150, Issue Suppl_1, Page AOr110-AOr110, November 12, 2024. Introduction:Lactic acidosis and impaired oxygen extraction due to mitochondrial dysfunction are common post-arrest. Thiamine, a cofactor for pyruvate dehydrogenase, is necessary for aerobic metabolism. In two randomized controlled trials (RCTs) testing the effect of thiamine vs. placebo in out-of-hospital and in-hospital post-arrest patients (NCT03450707 and NCT02974257), no relationship was found between thiamine treatment and the primary outcome of change in lactate over 24 hours. Cellular oxygen consumption rates (OCRs) were measured in a subset of patients at baseline. Maximal and spare OCRs measure the capacity of mitochondria to increase cellular respiration from their basal state when stimulated, and may identify patients likely to benefit from thiamine. We conducted a post-hoc analysis of the two RCTs to evaluate the primary outcome in subgroups defined by baseline OCRs.Hypothesis:Patients with higher maximal and spare OCRs at baseline are more likely to benefit from thiamine treatment, as indicated by lower lactate levels.Methods:Basal, maximal and spare OCRs, collected at enrollment, were measured in peripheral blood mononuclear cells using an XFe96 Extracellular Flux Analyzer and XF Cell Mito Stress Test Kit (Seahorse Bioscience). Lactates (at 6, 12, and 24 hours) were log-transformed and analyzed using a linear mixed model controlling for baseline lactate. In patients who expired

Circulation, Volume 150, Issue Suppl_1, Page A4119195-A4119195, November 12, 2024. Introduction:Acute myocardial infarction (AMI) is a leading global cause of mortality. Following primary percutaneous coronary intervention (PCI), 25% require hospitalisation for symptomatic heart failure. Further understanding of the molecular pathogenesis and the temporal relationship of the protein profile associated with ischaemic insult and subsequent cardiac remodelling is required to improve risk stratification following AMI.Methods:20 patients with AMI presenting to Royal Free Hospital in London were recruited for proteomic analysis (O link Proximity Extension Immunoassay) at presentation and at 24 and 72 hours following primary PCI. All underwent initial CMR on admission and at 6 month follow up to assess for myocardial oedema, left ventricular volumes and function and assessment of scar.Results:Proteomic biomarkers that increased on discharge following AMI were: IGFBP2 (8% increase, p=0.001), MMP-10 (5% increase, p=0.001), TIMP4 (14.4% increase, p

Circulation, Volume 150, Issue Suppl_1, Page A4143847-A4143847, November 12, 2024. Introduction:Our previous studies have shown that sustained activation of the DNA damage response (DDR) in cardiomyocytes leads to p53/p21 activation and cardiac dysfunction. Although the DDR generally involves molecules in DNA replication and repair pathways, the non-proliferative nature of cardiomyocytes suggests a cardio-specific DDR mechanism. However, our understanding of DDR in cardiomyocytes remains limited. Here, we aim to use CRISPR interference (CRISPRi) knockdown screens to identify genes critically involved in DDR regulation in human cardiomyocytes. We hypothesize that identifying these gene clusters may allow us to develop methods to prevent cardiac dysfunction by suppressing DDR in cardiomyocytes.Methods and Results:We established a human iPS cell line stably expressing dCas9-KRAB, which allows CRISPRi-mediated gene knockdown, and differentiated the cells into cardiomyocytes. The resulting human iPS cell-derived cardiomyocytes (hiPSCMs) showed the achievement of approximately 80% knockdown efficiency after gRNA transfection. We stimulated the hiPSCMs with H2O2and quantitatively evaluated the expression levels of the DDR markers γH2AX and p21 by immunostaining using the Operetta®high content imaging system. The DDR markers showed a significant concentration-dependent increase in response to H2O2administration. For arrayed CRISPRi screening, we constructed a gRNA library targeting 437 DDR-related genes. Using this library, we knocked down each DDR-related gene in hiPSCMs followed by H2O2stimulation. We quantified the expression levels of DDR markers by calculating the fluorescence intensity ratios relative to control after gene knockdown, and standardized them to calculate Z scores for all 437 genes. The screening successfully revealed the differential impact of each gene knockdown on γH2AX and p21 expression. We identified 71 genes that significantly affected their expression (Z-score < -1 or > 1). Mapping these genes to DDR pathways highlighted the differential impact of gene knockdown within the same pathway, and stratified their importance in cardiomyocytes.Conclusions:Arrayed CRISPR screening using hiPSCMs revealed differential functional significance of DDR-related genes in cardiomyocytes, identifying 71 genes of particularly significant importance. These findings provide a critical understanding of the cardio-specific DDR pathway and important clues for establishing an appropriate method to suppress DDR in the failing heart.

Circulation, Volume 150, Issue Suppl_1, Page A4143744-A4143744, November 12, 2024. Introduction/Background:Hypertension and hypercholesterolemia are important risk factors of endothelial dysfunction and atherosclerosis. Previous studies suggested a crosstalk between an activated renin-angiotensin-aldosterone system (RAAS), reactive oxygen species (ROS) and oxidized low-density lipoproteins (oxLDL) in atherosclerosis, but the underlying molecular mechanisms are not well understood.Research Question/Hypothesis:Can we identify novel signaling pathways controlling the molecular crosstalk of the RAAS with ROS and oxLDL in endothelial dysfunction and atherosclerosis?Methods/Approach:The impact of AT1R blockade on oxLDL-induced superoxide anion formation and endothelial dysfunction was studied in human umbilical artery endothelial cells and aortic rings of wild-type mice by chemiluminescence and Mulvany myograph. We cloned 5’-terminal deletions of the AT1R promoter and assessed the luciferase activity in human endothelial cells. Oct-1 binding to the human AT1R promoter region was studied by EMSA. Further assays included real-time PCR, confocal microscopy, Western blotting, G protein reporter assays, phospho-ERK1/2 antibodies and specific siRNAs. The data were validated in heart of obese C57BL/6 mice and cardiac and aortic tissue of AT1a/AT1bdouble knockout micein vivo.Results/Data:AT1R promoter activation studies upon Ang II- and oxLDL-stimulation in endothelial cells revealed that Ang II and oxLDL activate AT1R signaling through G protein Gα12/13, followed by activation of ERK1/2 MAP kinases, and transcription and translation of Oct-1, resulting in up-regulation of AT1R, LOX-1 and NOX2 expression, which could be antagonized by specific inhibitors at each step of the identified signaling cascade. AT1R blockade improved oxLDL-induced endothelial dysfunction in aortic rings of wild-type mice. Male C57BL/6 mice fed a high-fat diet exhibited upregulation of Oct-1 levels in cardiac tissues, compared to normal controls, while AT1a/AT1bdouble knockout mice demonstrated downregulation of Oct-1, AT1R, LOX-1, and NOX2 on mRNA and protein level in cardiac and aorta tissue, thus confirming the identified signaling cascadein vivo.Conclusions:Oct-1 is an essential transcription factor for Ang II- and oxLDL-induced upregulation of AT1R and LOX-1 expression in endothelium, thus identifying a novel molecular cross-talk of oxLDL with ROS signaling and the RAAS contributing to development of endothelial dysfunction and atherosclerosis.