Transverse Aortic Coarctation Research Articles

#977 Etelcalcetide improve left ventricular hypertrophy by suppressing the renin-angiotensin-aldosterone system and cardiac fibroblast growth factor 23

Abstract Background and Aims Calcimimetics are allosteric activators of the calcium-sensing receptor (CaSR) and are employed as therapeutic agents for managing secondary hyperparathyroidism in chronic kidney disease (CKD) patients undergoing dialysis. Although they have shown potential in reducing cardiovascular diseases and suppressing left ventricular hypertrophy (LVH), the mechanisms underlying these effects are not fully elucidated. In this study, we focused on exploring the mechanisms behind the suppression of LVH by calcimimetics using CKD, LVH, and CKD+LVH model rats. Specifically, we investigated the impact on CKD-mineral bone disorder (CKD-MBD) parameters, the renin-angiotensin-aldosterone system (RAAS), and calcineurin/nuclear factor of activated T cells (CaN/NFAT) pathway. Method In this study, we used etelcalcetide (ET) as a calcimimetic agent. At 8 weeks of age, LVH was induced in rats through transverse aortic coarctation, and CKD was induced by 5/6 nephrectomy at 9 and 10 weeks of age. The rats were divided into the four main groups: the sham group, the CKD group, the LVH group, and the CKD+LVH group. Each of the latter three groups were further subdivided into a vehicle-treated group and an ET-treated group for a duration of eight weeks. At 19 weeks of age, echocardiography was conducted on all rats prior to sacrifice. Subsequently, the rats were sacrificed, and blood and urine analyses, mRNA expression analysis, and renal histological analysis were performed. We evaluated LVH, CKD-MBD, RAAS, and CaN/NFAT pathways across these seven groups. Results In both the CKD and CKD+LVH groups, serum levels of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) were significantly elevated, and these elevations were mitigated by ET. However, in the LVH group, serum PTH and FGF23 levels were not elevated, and ET did not induce any changes. No significant alterations in serum levels of phosphorus, calcium, 25(OH)vitamin, and 1,25(OH)2vitaminD levels due to ET were observed in any of the groups. Interestingly, ET suppressed the increase in myocardial weight/body weight ratios and the area of cardiomyocytes not only in the CKD and CKD+LVH group but also in the LVH group, despite no changes in serum PTH and FGF23 levels. Regarding the RAAS, ET attenuated the increase in serum aldosterone levels, expressions of cardiac angiotensin-converting enzyme (ACE), cardiac angiotensinogen, and cardiac angiotensin II in the LVH group and the CKD+LVH group. The expression of cardiac FGF23, which increased in the LVH group and the CKD+LVH group, was decreased by ET. Moreover, there was a significant correlation between cardiac RAAS parameters and cardiac FGF23 expressions. No alteration was observed in the CaN/NFAT pathway in response to ET. Conclusion Our findings indicated that ET effectively suppressed LVH in the CKD group and the CKD+LVH group, and it was also effective in the LVH group, independent of changes in serum CKD-MBD parameters. This suggests that ET may exert its LVH-suppressing effects through the RAAS pathway and cardiac FGF23.

Aerobic Exercise Attenuates Pressure Overload-Induced Myocardial Remodeling and Myocardial Inflammation via Upregulating miR-574-3p in Mice.

Exercise training can promote cardiac rehabilitation, thereby reducing cardiovascular disease mortality and hospitalization rates. MicroRNAs (miRs) are closely related to heart disease, among which miR-574-3p plays an important role in myocardial remodeling, but its role in exercise-mediated cardioprotection is still unclear. A mouse myocardial hypertrophy model was established by transverse aortic coarctation, and a 4-week swimming exercise training was performed 1 week after the operation. After swimming training, echocardiography was used to evaluate cardiac function in mice, and histopathologic staining was used to detect cardiac hypertrophy, myocardial fibrosis, and cardiac inflammation. Quantitative real-time polymerase chain reaction was used to detect the expression levels of miR-574-3p and cardiac hypertrophy markers. Western blotting detected the IL-6 (interleukin-6)/JAK/STAT inflammatory signaling pathway. Echocardiography and histochemical staining found that aerobic exercise significantly improved pressure overload-induced myocardial hypertrophy (n=6), myocardial interstitial fibrosis (n=6), and cardiac inflammation (n=6). Quantitative real-time polymerase chain reaction detection showed that aerobic exercise upregulated the expression level of miR-574-3p (n=6). After specific knockdown of miR-574-3p in mouse hearts with adeno-associated virus 9 using cardiac troponin T promoter, we found that the protective effect of exercise training on the heart was significantly reversed. Echocardiography and histopathologic staining showed that inhibiting the expression of miR-574-3p could partially block the effects of aerobic exercise on cardiac function (n=6), cardiomyocyte cross-sectional area (n=6), and myocardial fibrosis (n=6). Western blotting and immunohistochemical staining showed that the inhibitory effects of aerobic exercise on the IL-6/JAK/STAT pathway and cardiac inflammation were partially abolished after miR-574-3p knockdown. Furthermore, we also found that miR-574-3p exerts cardioprotective effects in cardiomyocytes by targeting IL-6 (n=3). Aerobic exercise protects cardiac hypertrophy and inflammation induced by pressure overload by upregulating miR-574-3p and inhibiting the IL-6/JAK/STAT pathway.

Identification of diagnostic model in heart failure with myocardial fibrosis and conduction block by integrated gene co-expression network analysis

BackgroundDespite the advancements in heart failure(HF) research, the early diagnosis of HF continues to be a challenging issue in clinical practice. This study aims to investigate the genes related to myocardial fibrosis and conduction block, with the goal of developing a diagnostic model for early treatment of HF in patients.MethodThe gene expression profiles of GSE57345, GSE16499, and GSE9128 were obtained from the Gene Expression Omnibus (GEO) database. After merging the expression profile data and adjusting for batch effects, differentially expressed genes (DEGs) associated with conduction block and myocardial fibrosis were identified. Gene Ontology (GO) resources, Kyoto Encyclopedia of Genes and Genomes (KEGG) resources, and gene set enrichment analysis (GSEA) were utilized for functional enrichment analysis. A protein-protein interaction network (PPI) was constructed using a string database. Potential key genes were selected based on the bioinformatics information mentioned above. SVM and LASSO were employed to identify hub genes and construct the module associated with HF. The mRNA levels of TAC mice and external datasets (GSE141910 and GSE59867) are utilized for validating the diagnostic model. Additionally, the study explores the relationship between the diagnostic model and immune cell infiltration.ResultsA total of 395 genes exhibiting differential expression were identified. Functional enrichment analysis revealed that these specific genes primarily participate in biological processes and pathways associated with the constituents of the extracellular matrix (ECM), immune system processes, and inflammatory responses. We identified a diagnostic model consisting of 16 hub genes, and its predictive performance was validated using external data sets and a transverse aortic coarctation (TAC) mouse model. In addition, we observed significant differences in mRNA expression of 7 genes in the TAC mouse model. Interestingly, our study also unveiled a correlation between these model genes and immune cell infiltration.ConclusionsWe identified sixteen key genes associated with myocardial fibrosis and conduction block, as well as diagnostic models for heart failure. Our findings have significant implications for the intensive management of individuals with potential genetic variants associated with heart failure, especially in the context of advancing cell-targeted therapy for myocardial fibrosis.

Ovarian Tumor Domain-Containing 7B Attenuates Pathological Cardiac Hypertrophy by Inhibiting Ubiquitination and Degradation of Krüppel-Like Factor 4.

Cardiac hypertrophy (CH) is a well-established risk factor for many cardiovascular diseases and a primary cause of mortality and morbidity among older adults. Currently, no pharmacological interventions have been specifically tailored to treat CH. OTUD7B (ovarian tumor domain-containing 7B) is a member of theovarian tumor-related protease (OTU)family that regulates many important cell signaling pathways. However, the role of OTUD7B in the development of CH is unclear. Therefore, we investigated the role of OTUD7B in CH. OTUD7B knockout mice were used to assay the role of OTUD7B in CH after transverse aortic coarctation surgery. We further assayed the specific functions of OTUD7B in isolated neonatal rat cardiomyocytes. We found that OTUD7B expression decreased in hypertrophic mice hearts and phenylephrine-stimulated neonatal rat cardiomyocytes. Furthermore, OTUD7B deficiency exacerbated transverse aortic coarctation surgery-induced myocardial hypertrophy, abnormal cardiac function, and fibrosis. In cardiac myocytes, OTUD7B knockdown promoted phenylephrine stimulation-induced myocardial hypertrophy, whereas OTUD7B overexpression had the opposite effect. An immunoprecipitation-mass spectrometry analysis showed that OTUD7B directly binds to KLF4 (Krüppel-like factor 4). Additional molecular experiments showed that OTUD7B impedes KLF4 degradation by inhibiting lysine residue at 48 site-linked ubiquitination and suppressing myocardial hypertrophy by activating the serine/threonine kinase pathway. These results demonstrate that the OTUD7B-KLF4 axis is a novel molecular target for CH treatment.

Gpx3 and Egr1 Are Involved in Regulating the Differentiation Fate of Cardiac Fibroblasts under Pressure Overload.

Objectives Although myocardial fibrosis is a common pathophysiological process associated with many heart diseases, the molecular mechanisms regulating the development of fibrosis have not been fully determined. Recently, single cell RNA sequencing (scRNA-seq) analysis has been used to examine cellular fate and function during cellular differentiation and has contributed to elucidating the mechanisms of various diseases. The main purpose of this study was to characterize the fate of cardiac fibroblasts (CFs) and the dynamic gene expression patterns in a model of cardiac pressure overload using scRNA-seq analysis. Methods The public scRNA-seq dataset of the transverse aortic coarctation (TAC) model in mice was downloaded from the GEO database, GSE155882. First, we performed quality control, dimensionality reduction, clustering, and annotation of the data through the Seurat R package (v4.0.5). Then, we constructed the pseudotime trajectory of cell development and identified key regulatory genes using the Monocle R package (v2.22.0). Different cell fates and groups were fully characterized by Gene Set Enrichment Analysis (GSEA) analysis and Transcription factor (TF) activity analysis. Finally, we used Cytoscape (3.9.1) to extensively examine the gene regulatory network related to cell fate. Results Pseudotime analysis showed that CFs differentiated into two distinct cell fates, one of which produced activated myofibroblasts, and the other which produced protective cells that were associated with reduced fibrosis levels, increased antioxidative stress responses, and the ability to promote angiogenesis. In the TAC model, activated CFs were significantly upregulated, while protective cells were downregulated. Treatment with the bromodomain inhibitor JQ1 reversed this change and improved fibrosis. Analysis of dynamic gene expression revealed that Gpx3 was significantly upregulated during cell differentiation into protective cells. Gpx3 expression was affected by JQ1 treatment. Furthermore, Gpx3 expression levels were negatively correlated with the different levels of fibrosis observed in the various treatment groups. Finally, we found that transcription factors Jun, Fos, Atf3, and Egr1 were upregulated in protective cells, especially Egr1 was predicted to be involved in the regulation of genes related to antioxidant stress and angiogenesis, suggesting a role in promoting differentiation into this cell phenotype. Conclusions The scRNA-seq analysis was used to characterize the dynamic changes associated with fibroblast differentiation and identified Gpx3 as a factor that might be involved in the regulation of myocardial fibrosis under cardiac pressure overload. These findings will help to further understanding of the mechanism of fibrosis and provide potential intervention targets.

Chronic Renin-Angiotensin System Activation Induced Neuroinflammation: Common Mechanisms Underlying Hypertension and Dementia?

Hypertension is a major risk factor for the pathogenesis of vascular dementia and Alzheimer's disease. Chronic activation of the renin-angiotensin system (RAS) contributes substantially to neuroinflammation. We propose that neuroinflammation arising from chronic RAS activation can initiate and potentiate the onset of hypertension and related dementia. Neuroinflammation induced by chronic activation of the RAS plays a key role in the pathogenesis of dementia. Increased levels of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and transforming growth factor (TGF)-β have been reported in brain tissue of vascular dementia patients and animal models of vascular dementia induced by either angiotensin II infusion or transverse aortic coarctation. It is proposed that neuronal cell death and synaptic dysfunction induced by neuroinflammation lead to cognitive impairment in dementia. The neuroprotective RAS pathway, regulated by angiotensin-converting enzyme 2 (ACE2) which converts angiotensin II into angiotensin-(1-7), can attenuate hypertension and dementia. Furthermore, the use of anti-hypertensive medications in preventing dementia or cognitive decline in hypertensive patients and animal models of dementia have mostly been beneficial. Current evidence suggests a strong link between RAS induced neuroinflammation and the onset of hypertension and dementia, which warrants further investigation. Strategies to counteract an overactive RAS and enhance the neuroprotective arm of the RAS may help prevent or improve cognitive impairment associated with hypertension.

Tetralogy of Fallot and transverse aortic coarctation: A rare coexistence and its treatment.

Tetralogy of Fallot and transverse aortic coarctation: A rare coexistence and its treatment.

Ginsenoside Rg3 ameliorates myocardial glucose metabolism and insulin resistance via activating the AMPK signaling pathway

BackgroundGinsenoside Rg3 is one of the main active ingredients in ginseng. Here, we aimed to confirm its protective effect on the heart function in transverse aortic coarctation (TAC)-induced heart failure mice and explore the potential molecular mechanisms involved. MethodsThe effects of ginsenoside Rg3 on heart and mitochondrial function were investigated by treating TAC-induced heart failure in mice. The mechanism of ginsenoside Rg3 for improving heart and mitochondrial function in mice with heart failure was predicted through integrative analysis of the proteome and plasma metabolome. Glucose uptake and myocardial insulin sensitivity were evaluated using micro-positron emission tomography. The effect of ginsenoside Rg3 on myocardial insulin sensitivity was clarified by combining in vivo animal experiments and in vitro cell experiments. ResultsTreatment of TAC-induced mouse models with ginsenoside Rg3 significantly improved heart function and protected mitochondrial structure and function. Fusion of metabolomics, proteomics, and targeted metabolomics data showed that Rg3 regulated the glycolysis process, and Rg3 not only regulated glucose uptake but also improve myocardial insulin resistance. The molecular mechanism of ginsenoside Rg3 regulation of glucose metabolism was determined by exploring the interaction pathways of AMPK, insulin resistance, and glucose metabolism. The effect of ginsenoside Rg3 on the promotion of glucose uptake in IR-H9c2 cells by AMPK activation was dependent on the insulin signaling pathway. ConclusionsGinsenoside Rg3 modulates glucose metabolism and significantly ameliorates insulin resistance through activation of the AMPK pathway.

Sophoricoside ameliorates cardiac hypertrophy by activating AMPK/mTORC1-mediated autophagy.

Aim: The study aims to evaluate protective effects of sophoricoside (Sop) on cardiac hypertrophy. Meanwhile, the potential and significance of Sop should be broadened and it should be considered as an attractive drug for the treatment of pathological cardiac hypertrophy and heart failure.Methods: Using the phenylephrine (PE)-induced neonatal rat cardiomyocytes (NRCMs) enlargement model, the potent protection of Sop against cardiomyocytes enlargement was evaluated. The function of Sop was validated in mice received transverse aortic coarctation (TAC) or sham surgery. At 1 week after TAC surgery, mice were treated with Sop for the following 4 weeks, the hearts were harvested after echocardiography examination.Results: Our study revealed that Sop significantly mitigated TAC-induced heart dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis. Mechanistically, Sop treatment induced a remarkable activation of AMPK/mTORC1-autophagy cascade following sustained hypertrophic stimulation. Importantly, the protective effect of Sop was largely abolished by the AMPKα inhibitor Compound C, suggesting an AMPK activation-dependent manner of Sop function on suppressing pathological cardiac hypertrophy.Conclusion: Sop ameliorates cardiac hypertrophy by activating AMPK/mTORC1-mediated autophagy. Hence, Sop might be an attractive candidate for the treatment of pathological cardiac hypertrophy and heart failure.

Abstract 14930: Involvement of Chloride Channel (clic) in Myocardial Fibrosis of Pressure-overload Model Mice and Dilated Cardiomyopathy Patients

Introduction: Pressure overload induces cardiac hypertrophy, electrical remodeling due to changes in various channels, and eventually leads to fibrosis, hardening of the heart, left ventricular diastolic dysfunction, and heart failure. Chloride channels (chloride intracellular channels, Clic) are localized in both plasma membranes and intracellular organelles of various cells, and have recently been reported to be associated with atrial fibrosis. In this study, we examined single-cell RNA-seq analysis of cardiomyocytes for changes in Clic using a pressure overload model due to transverse aortic coarctation (TAC). In addition, a similar analysis was performed on ventricular myocytes in dilated cardiomyopathy. Methods: Single ventricular myocytes were collected from the free wall of the left ventricle after TAC by collagenase treatment in the cardiac hypertrophy phase (3 days after TAC, 1 or 2 weeks (W)) and the heart failure phase (4,8W). The expression of various genes in ventricular myocytes was analyzed for single cells by RNA-seq and compared with sham mice. In addition, a similar study was performed from single ventricular myocytes obtained from dilated cardiomyopathy patients and donors. Results: The expression of myosin heavy chain β (Myh7), a fetal gene, was increased by pressure overload, but the expression of BNP and ANP genes was increased along with the expression of adult Myh6. Regarding Clic, Clic1,4 and 5 increased as compared with Sham mice. Clic1, Clic1 and 4 increased from 3d, and at 8W, Clic1, 4, and 5 also increased . The expression level of these Clic genes is associated with the genes associated with fibrosis (Col4a1, 4a2, 6a2, connective tissue growth factor (CTGF), transforming growth factor-beta 2 (TGFβ2). The KEGG pathway analysis using DAVID for genes with significant associations with Clic1,4, and 5 revealed a strong association with activation of Focal adhesion pathway. Single cell RNA-seq analysis of single ventricular myocytes from patients with dilated cardiomyopathy also showed a increase in CLIC4 and 5 genes compared to healthy donors. Conclusions: Single cell RNA of ventricular myocytes -seq analysis revealed the involvement of chloride channel Clic in myocardial fibrosis and structural remodeling in failing hearts.

LncRNA NEAT1 promotes cardiac hypertrophy through microRNA-19a-3p/SMYD2 axis.

The role of NEAT1 in cancers has been demonstrated. But the role of NEAT1 in cardiac hypertrophy still remains unknown. This study aimed to elucidate the specific function of long non-coding RNA (lncRNA) NEAT1 in cardiac hypertrophy and its underlying mechanism. In this study, the in vivo and in vitro cardiac hypertrophy models were constructed by transverse aortic coarctation (TAC) procedure in rats and phenylephrine (PE) induction in primary cardiomyocytes, respectively. The expression levels of NEAT1, microRNA-19a-3p, SMYD2, and cardiac hypertrophic markers were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cardiac hypertrophy was evaluated as calculating the surface area of hypertrophic cardiomyocyte by fluorescein isothiocyanate (FITC)-Phalloidin staining. Luciferase Reporter Gene Assay was conducted to detect the binding of NEAT1, SMYD2, and microRNA-19a-3p. The results showed that NEAT1 and SMYD2 were highly expressed in myocardium of rats with cardiac hypertrophy and PE-induced primary cardiomyocytes, whereas microRNA-19a-3p was lowly expressed. Besides, NEAT1 overexpression markedly upregulated levels of the cardiac hypertrophic markers. Moreover, FITC-Phalloidin staining also revealed hypertrophic cardiomyocytes overexpressing NEAT1. On the contrary, microRNA-19a-3p overexpression reduced the cardiomyocyte surface area and downregulated the levels of the cardiac hypertrophic markers. As luciferase reporter gene assay demonstrated, NEAT1 and SMYD2 could bind to microRNA-19a-3p. Finally, the gain-of-function experiments were designed to verify whether NEAT1 exerted its functions in cardiac hypertrophy by modulating SMYD2 and microRNA-19a-3p. Furthermore, both microRNA-19a-3p overexpression or SMYD2 knockdown could inhibit and reduce the cardiomyocyte surface area, and downregulate the levels of the cardiac hypertrophic markers. In summary, NEAT1 promotes the occurrence and progression of cardiac hypertrophy by upregulating SMYD2 by binding to microRNA-19a-3p.

Tanshinol Borneol Ester Improves Pressure Overload-Induced Cardiac Hypertrophy Through Mitigating Autophagy

Autophagy plays an important role in pressure overload-induced cardiac hypertrophy. Tanshinol borneol ester (DBZ), which is a combination of Dan Shen abstract and borneol, has been proved to be effective in attenuating various cardiovascular diseases. However, whether the effect of DBZ in attenuating cardiac hypertrophy is through regulation of autophagy is unknown. In this study, we investigated the effect of DBZ both in vitro and in vivo. Neonatal rat ventricular myocytes (NRCMs) were cultured to test the effect of DBZ in vitro, and transverse aortic coarctation (TAC) was performed in rats for in vivo study. Results demonstrated that DBZ reduced the size of heart, genes and proteins expression of hypertrophic markers, ROS generation, cellular stress, remodeling and ameliorated the function of left ventricular. Moreover, autophagy was significantly decreased, indicating that DBZ may improve cardiac hypertrophy by inhibiting autophagy. Finally, our results showed that DBZ regulates autophagy via mTOR/p70S6K and ERK1/2 rather than the p38 pathway. In conclusion, these results suggest that DBZ can attenuate cardiac hypertrophy through inhibiting autophagy via mTOR/p70S6K and ERK1/2 pathway. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (81873493 to D.S.), Science and technology innovation personnel of the Education Department of Henan Province (19HASTIT002 to D.S.), Science and Technology Leading Talents of Health Commission of Henan Province (51282 project, 100 to D.S.) 5451 Project of Henan Provincial Commission of Health and Family Planning (2015002 to D.S.). Additional support from the University-College Joint Cultivation Fund of Zheng Zhou University (2016-BSTDJJ-19), Henan Province’s Key Laboratory of Cardiac Injury and Repair. Declaration of Interests: All authors have no competing interests to declare, financial or otherwise. Ethics Approval Statement: All experimental protocols were approved by the Animal Research Center of Zheng Zhou University and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996).

Alterations of Ocular Hemodynamics Impair Ophthalmic Vascular and Neuroretinal Function

Hypertension is associated with numerous diseases, but its direct impact on the ocular circulation and neuroretinal function remains unclear. Herein, mouse eyes were challenged with different levels of hemodynamic insult via transverse aortic coarctation, which increased blood pressure and flow velocity by 50% and 40%, respectively, in the right common carotid artery, and reduced those parameters by 30% and 40%, respectively, in the left common carotid artery. Blood velocity in the right central retinal artery gradually increased up to 40% at 4 weeks of transverse aortic coarctation, and the velocity in the left central retinal artery gradually decreased by 20%. The fundus and retinal architecture were unaltered by hemodynamic changes. Endothelium-dependent vasodilations to acetylcholine and adenosine were reduced only in right (hypertensive) ophthalmic arteries. Increased cellularity in the nerve fiber/ganglion cell layers, enhanced glial fibrillary acidic protein expression, and elevated superoxide level were found only in hypertensive retinas. The electroretinogram showed decreased scotopic b-waves in the hypertensive eyes and decreased scotopic oscillatory potentials in both hypertensive and hypotensive eyes. In conclusion, hypertension sustained for 4 weeks causes ophthalmic vascular dysfunction, retinal glial cell activation, oxidative stress, and neuroretinal impairment. Although ophthalmic vasoregulation is insensitive to hypotensive insult, the ocular hypoperfusion causes neuroretinal dysfunction.

Enhanced endothelin-1/Rho-kinase signalling and coronary microvascular dysfunction in hypertensive myocardial hypertrophy.

Hypertensive cardiac hypertrophy is associated with reduced coronary flow reserve, but its impact on coronary flow regulation and vasomotor function remains incompletely understood and requires further investigation. Left ventricular hypertrophy was induced in mice by transverse aortic coarctation (TAC) for 4 weeks. The left coronary artery blood velocity (LCABV) and myocardium lactate level were measured following the metabolic activation by isoproterenol. Septal coronary arterioles were isolated and pressurized for functional studies. In TAC mice, the heart-to-body weight ratio was increased by 45%, and cardiac fractional shortening and LCABV were decreased by 51 and 14%, respectively. The resting myocardial lactate level was 43% higher in TAC mice. Isoproterenol (5 µg/g, i.p.) increased heart rate by 20% in both groups of animals, but the corresponding increase in LCABV was not observed in TAC mice. The ventricular hypertrophy was associated with elevation of myocardial endothelin-1 (ET-1), increased vascular expression of rho-kinases (ROCKs), and increased superoxide production in the myocardium and vasculature. In coronary arterioles from TAC mice, the endothelial nitric oxide (NO)-mediated dilation to acetylcholine (ACh) was reversed to vasoconstriction and the vasoconstriction to ET-1 was augmented. Inhibition of ROCK by H-1152 alleviated oxidative stress and abolished enhanced vasoconstriction to ET-1. Both H-1152 and superoxide scavenger Tempol abolished coronary arteriolar constriction to ACh in a manner sensitive to NO synthase blocker NG-nitro-L-arginine methyl ester. Myocardial hypertrophy induced by pressure overload leads to cardiac and coronary microvascular dysfunction and ischaemia possibly due to oxidative stress, enhanced vasoconstriction to ET-1 and compromised endothelial NO function via elevated ROCK signalling.

Abstract 19425: A Small Molecule Enforcing MEF2 Deacetylation Prevents and Reverses Hypertrophy and Normalizes Cardiac Function in Multiple Stress Models

Background: Cardiac hypertrophy leads to progressive cardiac dysfunction, and is an independent risk factor for death. Myocyte enhancer factor 2 (MEF2) is silenced by interaction with class IIa histone deacetylases and activated by p300 acetyltransferase during hypertrophy. Hypothesis: Targeting the co-regulatory site on MEF2 will prevent and reverse hypertrophy and associated cardiac dysfunction. Methods: Transverse aortic coarctation (TAC) and swimming stress models were used to initiate cardiac hypertrophy in male C57Bl/6 mice. Mice swam for 3 hours twice daily; control mice were immersed briefly. 8MI was administered by daily IP injection at a dose of 40 mg/kg beginning either at the time of intervention (TAC, swimming stress) or 4 weeks afterward (TAC) and continued for 4 weeks. N ≥ 5 per group. RT-PCR, immunohistochemistry, and echocardiography were used to assess cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were treated with 8MI, the histone deacetylase inhibitors MC1568 (class IIa-selective) or trichostatin A (nonselective, TSA). Results: Both TAC and forced swimming induced pathological hypertrophy with deterioration in systolic function in mice receiving DMSO (p<0.001 for both). 4 weeks after TAC or the initiation of swimming, 8MI-treated animals had myocardial mass and function similar to those of sham-operated or immersed-control mice. After 8MI treatment was stopped at 4 weeks, hypertrophy developed in mice that continued to swim, similar to DMSO-treated animals. In mice with established hypertrophy 4 weeks after TAC, treatment with 8MI, but not DMSO, reversed systolic dysfunction (n=5 per group, p=0.0018) and reduced myocardial wall thickness to near that of sham-operated animals. No evidence of systemic toxicity was seen. Mechanistically, 8MI, but not MC1568, promoted HDAC4 nuclear retention and maintained MEF2 in a deacetylated state, and unlike TSA, prevented EP300 accumulation. Conclusions: Pharmacological targeting of MEF acetylation in vivo prevents and reverses stress-associated hypertrophy in multiple models. Reversal of hypertrophy by this means improves systolic function and does not prevent successful adaptation to increased cardiac work.

Cardiac hypertrophy leads to coronary microvascular dysfunction and enhanced endothelin‐1/Rho‐kinase signaling in mice with transverse aortic coarctation (676.1)

Hypertensive cardiac hypertrophy induced by transverse aortic coarctation (TAC) has been reported to reduce coronary flow reserve in mice, but its impact on oxygen delivery and microvascular function remains unknown. In the present study, we found that induction of TAC in mice for 3‐4 weeks increased heart‐to‐body weight ratio by 55% and decreased cardiac fractional shortening and left coronary arterial blood velocity (LCABV) by 51% and 21%, respectively. The myocardial lactate level was 43% higher in TAC than sham control mice. Isoproterenol (5 µg/g, i.p.) increased heart rate by 20% but the increased LCABV (23%) was observed only in the sham mice. In TAC mice, the superoxide levels in myocardium and coronary arterioles were increased by 1.7‐ and 2.3‐fold, respectively, and the myocardial endothelin‐1 (ET‐1) was increased by 59%. The endothelium‐dependent, nitric oxide (NO)‐mediated vasodilations to acetylcholine and adenosine, but not endothelium‐independent vasodilations to NO donor sodium nitroprusside, were impaired in TAC mice. The Rho kinase (ROCK)‐dependent vasoconstriction to ET‐1 was enhanced and the expressions of ROCK‐1 and ‐2 were increased in coronary arterioles of TAC mice. We conclude that myocardial pressure overload leads to cardiac deficiency, coronary microvascular dysfunction and ischemia possibly due to oxidative stress, elevated ET‐1/ROCK signaling and compromised NO function.Grant Funding Source: Supported by Kruse Endowed Research Fund from Baylor Scott & White Healthcare

Abstract 18905: Myocyte-Specific Deletion of P300 Prevents Cardiac Hypertrophy Following Pressure Overload in a Mouse Model

Background: The lysine acetyltransferases CREB binding protein (CBP, KAT3A) and p300 (KAT3B) are important epigenetic regulators of gene transcription. Both share extensive sequence homology and multiple overlapping functions and properties. However, genetic loss-of-function studies indicate that CBP and p300 have distinct roles in cardiac development. We have previously shown that p300 is a stress-responsive, quantitatively limiting driver of cardiac hypertrophy. Objective: To compare the roles of p300 and CBP in adult cardiac hypertrophy. Methods: Recombination was induced in mice harboring the a-MHC Mer-Cre-Mer transgene and CBP or p300 floxed alleles with tamoxifen (TA) at 10 weeks of age. Pressure overload was induced by transverse aortic coarctation (TAC) and pressure gradients were quantitated by Doppler flow/velocity. Cardiac function was evaluated using echocardiography before and after surgery. Apoptosis and fibrosis was assessed using CardioTACs assay and Masson tri-chrome staining respectively. Myocyte hypertrophy and fibrosis were quantitated by histology at d21 post TAC. Genome-wide transcriptional analysis was performed by microarray and RNASeq. Results: TAC produced myocyte hypertrophy, cardiac enlargement, and systolic dysfunction that was reduced to near sham-operated levels in a gene-dose-dependent manner by loss of p300 (p<0.001 for p300 fl/fl vs p300+/+). Loss of p300 did not impair survival post TAC. Histologically, fibrosis and myocyte apoptosis after TAC were markedly decreased in p300fl/fl vs p300+/+ mice (15.04% vs 38.92%,p<0.001). Remarkably, cardiac systolic function (EF % and FS %) were unimpaired in p300KO mice following TAC. Loss of CBP had no impact on any of these parameters. Transcriptional analysis revealed that loss of CBP had a much greater impact on gene expression than loss of p300, and that relatively few genes were responsive to both p300 and CBP loss. A group of 89 mRNAs and ncRNAs were exclusively sensitive to p300 loss, reflecting an important function in oxidative stress. Conclusions: Cardiac myocyte p300 plays a critical and cell-autonomous role in cardiac hypertrophy and heart failure that is not complemented by CBP. Investigation of p300 specific gene targets is warranted.

Macrophage migration inhibitory factor antagonizes pressure overload-induced cardiac hypertrophy

Macrophage migration inhibitory factor (MIF) functions as a proinflammatory cytokine when secreted from the cell, but it also exhibits antioxidant properties by virtue of its intrinsic oxidoreductase activity. Since increased production of ROS is implicated in the development of left ventricular hypertrophy, we hypothesized that the redox activity of MIF protects the myocardium when exposed to hemodynamic stress. In a mouse model of myocardial hypertrophy induced by transverse aortic coarctation (TAC) for 10 days, we showed that growth of the MIF-deficient heart was significantly greater by 32% compared with wild-type (WT) TAC hearts and that fibrosis was increased by fourfold (2.62 ± 0.2% vs. 0.6 ± 0.1%). Circulating MIF was increased in TAC animals, and expression of MIF receptor, CD74, was increased in the hypertrophic myocardium. Gene expression analysis showed a 10-fold increase (P < 0.01) in ROS-generating mitochondrial NADPH oxidase and 2- to 3-fold reductions (P < 0.01) in mitochondrial SOD2 and mitochondrial aconitase activities, indicating enhanced oxidative injury in the hypertrophied MIF-deficient ventricle. Hypertrophic signaling pathways showed that phosphorylation of cytosolic glycogen synthase kinase-3α was greater (P < 0.05) at baseline in MIF-deficient hearts than in WT hearts and remained elevated after 10-day TAC. In the hemodynamically stressed MIF-deficient heart, nuclear p21(CIP1) increased sevenfold (P < 0.01), and the cytosolic increase of phospho-p21(CIP1) was significantly greater than in WT TAC hearts. We conclude that MIF antagonizes myocardial hypertrophy and fibrosis in response to hemodynamic stress by maintaining a redox homeostatic phenotype and attenuating stress-induced activation of hypertrophic signaling pathways.

Hypertension Induces Brain β-Amyloid Accumulation, Cognitive Impairment, and Memory Deterioration Through Activation of Receptor for Advanced Glycation End Products in Brain Vasculature

Although epidemiological data associate hypertension with a strong predisposition to develop Alzheimer disease, no mechanistic explanation exists so far. We developed a model of hypertension, obtained by transverse aortic constriction, leading to alterations typical of Alzheimer disease, such as amyloid plaques, neuroinflammation, blood-brain barrier dysfunction, and cognitive impairment, shown here for the first time. The aim of this work was to investigate the mechanisms involved in Alzheimer disease of hypertensive mice. We focused on receptor for advanced glycation end products (RAGE) that critically regulates Aβ transport at the blood-brain barrier and could be influenced by vascular factors. The hypertensive challenge had an early and sustained effect on RAGE upregulation in brain vessels of the cortex and hippocampus. Interestingly, RAGE inhibition protected from hypertension-induced Alzheimer pathology, as showed by rescue from cognitive impairment and parenchymal Aβ deposition. The increased RAGE expression in transverse aortic coarctation mice was induced by increased circulating advanced glycation end products and sustained by their later deposition in brain vessels. Interestingly, a daily treatment with an advanced glycation end product inhibitor or antioxidant prevented the development of Alzheimer traits. So far, Alzheimer pathology in experimental animal models has been recognized using only transgenic mice overexpressing amyloid precursor. This is the first study demonstrating that a chronic vascular insult can activate brain vascular RAGE, favoring parenchymal Aβ deposition and the onset of cognitive deterioration. Overall we demonstrate that RAGE activation in brain vessels is a crucial pathogenetic event in hypertension-induced Alzheimer disease, suggesting that inhibiting this target can limit the onset of vascular-related Alzheimer disease.

Abstract P249: Cardiac Troponin I Pro82Ser Variant Impairs Myofilament Cooperativity, Induces Diastolic Dysfunction, and Blunts β-Adrenergic Response

Cardiac troponin I is a key regulatory protein for muscle contraction and necessary for an adequate β-adrenergic response. The Proline 82 Serine (P82S) sequence variant of troponin I gene ( TNNI3 ) is associated with late onset hypertrophic cardiomyopathy (HCM). Our preliminary data has shown that cTnIP82S is heterozygous in 3% of African Americans and is associated with increased LV mass in hypertensive black men. We hypothesize that cTnIP82S variant could influence the hypertrophic response to hypertension and/or the malignancy of the phenotypic expression when combined with known HCM disease causing myofilament mutations. We created a transgenic cTnIP82S mouse model. Echocardiography at baseline in older Tg and NTg mice (64-65 wks) showed impaired ejection time and relaxation, Tg display longer isovolumetric relaxation time (IVRT) (Tg 29.7±0.26 vs NTg 23± 0.23 msec, n=8 vs n=7 p&lt;0.05) and a significantly higher TEI index. In the young NTg and Tg (17 wks) there were no echocardiographic differences. However, preliminary data suggest that 1 wk after transverse aortic coarctation (TAC), diastolic dysfunction was evident in Tg, and 10 weeks after TAC LV mass was also increased compared to NTg. Skinned fibers studies showed in the Tg mice borderline lower maximal Force (F max ) and myofilament calcium sensitivity, whereas n Hill showed a marked depression (Tg 1.72±0.17 vs NTg 3.09±0.44, n=7 vs n=5, p&lt;0.05), this effect was not due to the global myofilament phosphorylation pattern. In addition, intact twitching cardiac muscle studies in Tg mice revealed an impaired dose-dependent β-adrenergic acceleration of relaxation, evidenced by a failure to increase relaxation ([-dF/dt min]/[+dF/dt max]) and accelerate calcium transients decay after isoproterenol. Pressure-volume (PV) loop studies confirm that Tg mice fail to increase: Δ change in dP/dt max (NTg 4,928.3±7.7 vs Tg 843.3±10.7, n=4 vs n=4 p&lt;0.05) in response to isoproterenol. TnIP82S variant is near a region of TnI-TnT interaction; this could explain its dramatic effects on myofilament cooperativity. Overall these studies suggest that the expression of cTnIP82S variant in the mice heart induces diastolic dysfunction, impairs relaxation at baseline and after β-adrenergic stimulation.