Cardiac Gene Expression Profile Research Articles

Exploring Ferroptosis-Associated Gene Signatures as Diagnostic and Therapeutic Targets for Sepsis-Induced Cardiomyopathy.

Sepsis-induced cardiomyopathy (SICM) is a severe complication of sepsis associated with high mortality rates. Despite its significance, the molecular mechanisms underlying SICM remain poorly understood, particularly the role of ferroptosis - a form of iron-dependent programmed cell death. This study analyzed the GSE79962 dataset from the Gene Expression Omnibus, containing cardiac gene expression profiles from SICM patients and controls. A list of ferroptosis-related genes (FRGs) was retrieved from the FerrDb. We used the limmapackage in R for differential expression analysis, setting an adjusted P-value cutoff of <0.05 and a log2-fold change threshold of ±1 to identify differentially expressed ferroptosis-related genes (DE-FRGs). We applied machine learning algorithmsfor biomarker identification, including least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine with recursive feature elimination (SVM-RFE), implemented via the glmnetand e1071packages in R, respectively. Gene set enrichment analysis (GSEA) was conducted using the GSEApackage to investigate the biological pathways related to key DE-FRGs. After differential expression analysis, we identified 145 DE-FRGs. Functional enrichment analyses underscored the involvement of these genes in critical biological processes and pathways, such as lipid metabolism and insulin resistance. Machine learning approaches pinpointed five key DE-FRGs (NCOA4, GABARAPL1, GJA1, CISD1, CP), with strong predictive potential for SICM. Further analyses, including the construction of a ceRNA network, revealed intricate post-transcriptional regulatory mechanisms that may influence the expression of these key genes. Our findings highlight the central role of ferroptosis in SICM and identify potential biomarkers and therapeutic targets that could help refine diagnostic and treatment strategies. This study advances our understanding of the molecular underpinnings of SICM and sets the stage for future research aimed at mitigating this severe sepsis complication.

Correlation of blood-based immune molecules with cardiac gene expression profiles reveals insights into Chagas cardiomyopathy pathogenesis.

Understanding compartmentalized immune responses in target organs is crucial for elucidating the pathogenesis of various diseases. However, obtaining samples from affected vital organs often poses safety challenges. In this study, we aimed to investigate potential correlations between the levels of disease-associated immune molecules in the bloodstream with their gene expression profiles in the hearts of patients suffering from Chagas Cardiomyopathy (CCC). This debilitating and often fatal condition is caused by infection with the protozoan Trypanosoma cruzi. Blood samples were analyzed using the Bio-Plex platform. Gene Expression Omnibus (GEO) database was used to determine gene expression profile in heart tissue from CCC and non-Chagas controls (CTRL). Elevated levels of inflammatory cytokines were detected in the plasma of CCC patients, and these levels correlated with clinical indicators of deteriorating cardiac function. Notably, 75% of the soluble factors assessed in the plasma exhibited a consistent relationship with their gene expression levels in the cardiac tissue of CCC patients. Analysis of interactions and signaling pathways related to these molecules revealed an overrepresentation of inflammatory pathways in both blood and heart compartments. Moreover, we identified that differentially expressed genes in CCC cardiac tissue were primarily associated with T-cell signaling pathways and correlated with the presence of CD8+ T cells in the myocardium. Our findings establish a strong correlation between relevant immune molecules and their signaling pathways in both the blood and heart tissue in CCC. This validates the use of blood as a non-invasive medium for understanding immunopathology and identifying markers for cardiac dysfunction in Chagas disease.

Characterizing Early Cardiac Metabolic Programming via 30% Maternal Nutrient Reduction during Fetal Development in a Non-Human Primate Model.

Intra-uterine growth restriction (IUGR) is a common cause of fetal/neonatal morbidity and mortality and is associated with increased offspring predisposition for cardiovascular disease (CVD) development. Mitochondria are essential organelles in maintaining cardiac function, and thus, fetal cardiac mitochondria could be responsive to the IUGR environment. In this study, we investigated whether in utero fetal cardiac mitochondrial programming can be detectable in an early stage of IUGR pregnancy. Using a well-established nonhuman IUGR primate model, we induced IUGR by reducing by 30% the maternal diet (MNR), both in males (MNR-M) and in female (MNR-F) fetuses. Fetal cardiac left ventricle (LV) tissue and blood were collected at 90 days of gestation (0.5 gestation, 0.5 G). Blood biochemical parameters were determined and heart LV mitochondrial biology assessed. MNR fetus biochemical blood parameters confirm an early fetal response to MNR. In addition, we show that in utero cardiac mitochondrial MNR adaptations are already detectable at this early stage, in a sex-divergent way. MNR induced alterations in the cardiac gene expression of oxidative phosphorylation (OXPHOS) subunits (mostly for complex-I, III, and ATP synthase), along with increased protein content for complex-I, -III, and -IV subunits only for MNR-M in comparison with male controls, highlight the fetal cardiac sex-divergent response to MNR. At this fetal stage, no major alterations were detected in mitochondrial DNA copy number nor markers for oxidative stress. This study shows that in 90-day nonhuman primate fetuses, a 30% decrease in maternal nutrition generated early in utero adaptations in fetal blood biochemical parameters and sex-specific alterations in cardiac left ventricle gene and protein expression profiles, affecting predominantly OXPHOS subunits. Since the OXPHOS system is determinant for energy production in mitochondria, our findings suggest that these early IUGR-induced mitochondrial adaptations play a role in offspring's mitochondrial dysfunction and can increase predisposition to CVD in a sex-specific way.

Effect of preventive training in cardiac transcriptoma in rats with pulmonary hypertension

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Pulmonary hypertension (PH) is a progressive disease characterized by elevated right ventricular afterload, vascular remodeling, right ventricular hypertrophy and dysfunction, and ultimately heart failure. Physical exercise has been recognized as a treatment for this condition. Although different mechanisms have been proposed to explain PH development, the influence of preventive exercise training interventions onset of pulmonary hypertension in right ventricular hypertrophy is underexplored. Purpose Analyze the effect of preventive training in global gene expression profiling of right ventricular in PH from rats. Methods Twenty-four male Wistar rats (206-220 g) were divided into three four groups (n=8/group): S, sedentary control; SPH, sedentary pulmonary hypertension; and TPH, trained pulmonary hypertension group. The preventive training protocol was performed on a treadmill for 13 weeks, five times/week. Cardiac hypertrophy by PH was experimentally induced by a single intraperitoneal injection (60 mg/kg) of monocrotaline after the protocol training. The S were injected with saline solution. The rats were evaluated 28 days after monocrotaline administration, in the early stage of the PH. Left ventricle weight, right ventricle weight, and atrium weight normalized by body weight were used as indexes of heart hypertrophy. Gene expression profiling was performed using the Rat Gene ST Array platform. To further understand the biological relevance of differential expressed genes, we performed a functional enrichment analysis in the context of the gene ontology (biological process, molecular function, and cellular component) and interaction networks (KEGG, Reactome, Wikipathways and BioPlanet). Results Through the analysis, the HP group had cardiac hypertrophy, and the training mitigated this change p&amp;lt; 0.05. Global gene expression profiling identified 687 differentially expressed genes (S vs. SPH), in the hypertrophy right ventricular during PH, and when compared to preventive training, 91 genes were dysregulated (SPH vs TPH). Gene ontology analysis on expression profiling data revealed alteration on genes that regulate the inflammatory process, muscle growth, metabolic processes, cell death, and ion regulation comparison between SPH vs. TPH The genes that were altered in the hypertrophy phase of PH and modified by the exercise were platelet alpha granule lumen (genes Fgg and Qsox1), NAD + binding (gene Cryl1), positive regulation of cAMP-mediated signaling (gene Cxcl11), positive regulation of macrophage differentiation (Il34 gene), mRNA cleavage (Cstf3 gene), negative regulation of transporter activity (Wwp2 gene), regulation of respiratory system process (Mtg2 gene) p&amp;lt; 0.05. Conclusion Preventive training influences changes in cardiac gene expression profile in the hypertrophy stage of PH. New genes have been identified, and these could be biomarkers and potential therapeutic targets in the early stage of PH.

EZH2 epigenetic enzyme inhibition improves the lipid metabolic disturbances and preserves cardiac function in a mouse model of myocardial infarction

EZH2 is an epigenetic enzyme repressing a wide variety of gene expression by adding methyl groups on histone H3 lysine 27 residue. In myocardial infarction (MI), most of these targeted genes are related to metabolism. This study aims to assess the benefit of GSK-343 (EZH2 inhibitor) on cardiac function and lipid metabolism in heart failure with reduced ejection fraction (HFrEF) induced by MI. We hypothesize that EZH2 modulates fatty acid (FA) cardiac metabolism and its inhibition minimizes lipid metabolism disruptions and improves cardiac function. Female mice underwent MI by ligation of left anterior descending coronary artery and were treated daily with vehicle or GSK-343 (7 days). We evaluated cardiac function (echocardiography), cardiac gene expression profiling (RNA-seq, RT-qPCR) and the circulating lipidome was assessed (mass spectrometry-based untargeted lipidomics). MI induced a reduction of EF from 46% (sham) to 18% (MI) while GSK-343 prevents this dysfunction (EF∼38%). RNA-seq analysis revealed wide gene expression changes in MI, changes normalized with GSK-343 treatment. The latter changes include, among the 561 down-regulated genes (FC 0.5 and 2; P = 10-4), 16 mitochondrial DNA-encoded genes (e.g. mt-Nd1, Mt-Rnr1), 57 genes related to mitochondrial function (e.g. Ppargc1-alpha) and 15 to lipid metabolism (e.g. Ppar-alpha, Acadm). As expected, most of these metabolic gene expressions were validated using RT-qPCR and emphasized a rescue upon GSK-343 treatment. We observed a similar pattern for other metabolic genes such as Klf15, an Ezh2 putative direct target gene and a master metabolic gene regulator, Cpt2 encoding a FA transporter and Pnpla2 implicated in triglyceride (TG) metabolism. Because disruption in FA utilization affect the lipidome, we used an untargeted lipidomics approach to evaluate the global impact of MI ± GSK-343. In plasma, while MI enhanced 18 TG (1.24–2.43-fold; P < 0.05) and decreased 32 choline glycerophospholipids (PC; 0.55–0.81-fold, P < 0.05), GSK-343 normalized these perturbations. Finally, in MI, some of these lipids positively correlated with EF (e.g. PC40:6; R = 0.84, P = 0.004) or with cardiac hypertrophy (e.g. DG18:1_18:2; R = 0.81, P = 0.007) while GSK-343 treatment abolished most of these correlations. Our study suggests that inhibiting EZH2 is of therapeutic interest to normalize lipid metabolism perturbations and improve cardiac function in HFrEF induced by MI.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obesity

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Emerging evidence suggests development of heart failure with preserved ejection fraction in females with CMD and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the hypothesis that smooth muscle cell (SMC)-specific MR deletion prevents obesity-associated coronary and cardiac diastolic dysfunction in females. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Initial studies revealed that global MR blockade with spironolactone prevented impaired coronary vasodilation and diastolic dysfunction in obese females. Importantly, specific deletion of SMC-MR similarly prevented obesity-associated coronary and cardiac dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in multiple non-myocyte populations (B/T cells, macrophages, and endothelium), independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling broadly impacting the cardiac cellulome and underlying obesity-associated coronary and cardiac dysfunction thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity. Supported by NIH R01 HL136386 (Bender), NIH R01 HL119290 (Jaffe), the University of Missouri Research Core Facilities Grant Program (Bender), NHMRC Ideas Grant GNT1188503 (Pinto), VA Merit I01 BX004220 and RCS IK2 6004016 (Chandrasekar). The work was also supported by resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.

Gene-Edited Human-Induced Pluripotent Stem Cell Lines to Elucidate DAND5 Function throughout Cardiac Differentiation.

(1) Background: The contribution of gene-specific variants for congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 on human cardiomyocyte (CM) differentiation and proliferation. (2) Methods: Taking advantage of our DAND5 patient-derived iPSC line, we used CRISPR-Cas9 gene-editing to generate a set of isogenic hiPSCs (DAND5-corrected and DAND5 full-mutant). The hiPSCs were differentiated into CMs, and RT-qPCR and immunofluorescence profiled the expression of cardiac markers. Cardiomyocyte proliferation was analysed by flow cytometry. Furthermore, we used a multi-electrode array (MEA) to study the functional electrophysiology of DAND5 hiPSC-CMs. (3) Results: The results indicated that hiPSC-CM proliferation is affected by DAND5 levels. Cardiomyocytes derived from a DAND5 full-mutant hiPSC line are more proliferative when compared with gene-corrected hiPSC-CMs. Moreover, parallel cardiac differentiations showed a differential cardiac gene expression profile, with upregulated cardiac progenitor markers in DAND5-KO hiPSC-CMs. Microelectrode array (MEA) measurements demonstrated that DAND5-KO hiPSC-CMs showed prolonged field potential duration and increased spontaneous beating rates. In addition, conduction velocity is reduced in the monolayers of hiPSC-CMs with full-mutant genotype. (4) Conclusions: The absence of DAND5 sustains the proliferation of hiPSC-CMs, which alters their electrophysiological maturation properties. These results using DAND5 hiPSC-CMs consolidate the findings of the in vitro and in vivo mouse models, now in a translational perspective. Altogether, the data will help elucidate the molecular mechanism underlying this human heart disease and potentiates new therapies for treating adult CHD.

Glucagon-like Peptide-1 receptor Tie2+ cells are essential for the cardioprotective actions of liraglutide in mice with experimental myocardial infarction

ObjectivesGlucagon-like peptide-1 receptor (GLP-1R) agonists reduce the rates of major cardiovascular events, including myocardial infarction in people with type 2 diabetes, and decrease infarct size while preserving ventricular function in preclinical studies. Nevertheless, the precise cellular sites of GLP-1R expression that mediate the cardioprotective actions of GLP-1 in the setting of ischemic cardiac injury are uncertain. MethodsPublicly available single cell RNA sequencing (scRNA-seq) datasets on mouse and human heart cells were analyzed for Glp1r/GLP1R expression. Fluorescent activated cell sorting was used to localize Glp1r expression in cell populations from the mouse heart. The importance of endothelial and hematopoietic cells for the cardioprotective response to liraglutide in the setting of acute myocardial infarction (MI) was determined by inactivating the Glp1r in Tie2+ cell populations. Cardiac gene expression profiles regulated by liraglutide were examined using RNA-seq to interrogate mouse atria and both infarcted and non-infarcted ventricular tissue after acute coronary artery ligation. ResultsIn mice, cardiac Glp1r mRNA transcripts were exclusively detected in endocardial cells by scRNA-seq. In contrast, analysis of human heart by scRNA-seq localized GLP1R mRNA transcripts to populations of atrial and ventricular cardiomyocytes. Moreover, very low levels of GIPR, GCGR and GLP2R mRNA transcripts were detected in the human heart. Cell sorting and RNA analyses detected cardiac Glp1r expression in endothelial cells (ECs) within the atria and ventricle in the ischemic and non-ischemic mouse heart. Transcriptional responses to liraglutide administration were not evident in wild type mouse ventricles following acute MI, however liraglutide differentially regulated genes important for inflammation, cardiac repair, cell proliferation, and angiogenesis in the left atrium, while reducing circulating levels of IL-6 and KC/GRO within hours of acute MI. Inactivation of the Glp1r within the Tie2+ cell expression domain encompassing ECs revealed normal cardiac structure and function, glucose homeostasis and body weight in Glp1rTie2−/− mice. Nevertheless, the cardioprotective actions of liraglutide to reduce infarct size, augment ejection fraction, and improve survival after experimental myocardial infarction (MI), were attenuated in Glp1rTie2−/− mice. ConclusionsThese findings identify the importance of the murine Tie2+ endothelial cell GLP-1R as a target for the cardioprotective actions of GLP-1R agonists and support the importance of the atrial and ventricular endocardial GLP-1R as key sites of GLP-1 action in the ischemic mouse heart. Hitherto unexplored species-specific differences in cardiac GLP-1R expression challenge the exclusive use of mouse models for understanding the mechanisms of GLP-1 action in the normal and ischemic human heart.

Abstract 14892: Interaction of β IV -Spectrin/STAT3 is Responsible for Orienting Concentric versus Eccentric Cardiomyocyte Hypertrophy

Cardiac hypertrophy is critical in the development of heart failure following chronic cardiac stress. In pressure overload, hypertrophy is divided by early adaptive remodeling defined by concentric growth, while chronic hypertrophy associates with eccentric growth and loss in cardiac output. The mechanisms regulating this transition and the impact each profile has in influencing heart function are largely unknown. Previous work identified a role for the structural protein βIV-spectrin and its interaction with the transcription factor, STAT3, in coordinating hypertrophic profiles. Specifically, mice expressing truncated βIV-spectrin lacking interaction with the hypertrophic signaling molecule CaMKII (qv 3J mice) were resistant to βIV-spectrin degradation and maintained concentric hypertrophy and ejection fraction (EF) compared to WT following 6 weeks transaortic constriction (TAC). Alternatively, mutant βIV-spectrin mice (qv 4J ) lacking the STAT3/βIV-spectrin binding domain lead to cytosolic redistribution of STAT3 and associated with reduced EF and LV dilation consistent in eccentric remodeling, suggesting STAT3/βIV-spectrin is a key regulator of hypertrophic profiles and function. This relationship was further examined by adenoviral overexpression of the hypertrophic driver, MEK1/2, on isolated myocytes. As expected, MEK increased the width of WT myocytes, while qv 4J myocytes increased in length. However, application of STAT3 inhibitor S3I-201 on qv 4J myocytes reoriented these cells to growth in width. Additionally, in vivo application of S3I-201 to qv 4J mice was able to restore LV wall thickness and EF over 2 weeks, reinforcing STAT3 as a determinant of hypertrophic profiles. Evaluation of cardiac gene expression profiles from these backgrounds identified consistent elevations in microtubule subunits associated with eccentric growth patterns. Indeed, microtubule growth assays revealed qv 4J myocytes to express significantly enhanced microtubule stability which could be reversed through S3I-201 STAT3 inhibition. Together, these data identify a novel role for βIV-spectrin/STAT3 and microtubule stability in tuning specific hypertrophic profiles and cardiac function.

Pro-oxidative priming but maintained cardiac function in a broad spectrum of murine models of chronic kidney disease

AimsPatients with chronic kidney disease (CKD) have an increased risk of cardiovascular events and exhibit myocardial changes including left ventricular (LV) hypertrophy and fibrosis, overall referred to as ‘uremic cardiomyopathy’. Although different CKD animal models have been studied for cardiac effects, lack of consistent reporting on cardiac function and pathology complicates clear comparison of these models. Therefore, this study aimed at a systematic and comprehensive comparison of cardiac function and cardiac pathophysiological characteristics in eight different CKD models and mouse strains, with a main focus on adenine-induced CKD. Methods and resultsCKD of different severity and duration was induced by subtotal nephrectomy or adenine-rich diet in various strains (C57BL/6J, C57BL/6 N, hyperlipidemic C57BL/6J ApoE−/−, 129/Sv), followed by the analysis of kidney function and morphology, blood pressure, cardiac function, cardiac hypertrophy, fibrosis, myocardial calcification and inflammation using functional, histological and molecular techniques, including cardiac gene expression profiling supplemented by oxidative stress analysis. Intriguingly, despite uremia of variable degree, neither cardiac dysfunction, hypertrophy nor interstitial fibrosis were observed. However, already moderate CKD altered cardiac oxidative stress responses and enhanced oxidative stress markers in each mouse strain, with cardiac RNA sequencing revealing activation of oxidative stress signaling as well as anti-inflammatory feedback responses. ConclusionThis study considerably expands the knowledge on strain- and protocol-specific differences in the field of cardiorenal research and reveals that several weeks of at least moderate experimental CKD increase oxidative stress responses in the heart in a broad spectrum of mouse models. However, this was insufficient to induce relevant systolic or diastolic dysfunction, suggesting that additional “hits” are required to induce uremic cardiomyopathy. Translational perspectivePatients with chronic kidney disease (CKD) have an increased risk of cardiovascular adverse events and exhibit myocardial changes, overall referred to as ‘uremic cardiomyopathy’. We revealed that CKD increases cardiac oxidative stress responses in the heart. Nonetheless, several weeks of at least moderate experimental CKD do not necessarily trigger cardiac dysfunction and remodeling, suggesting that additional “hits” are required to induce uremic cardiomyopathy in the clinical setting. Whether the altered cardiac oxidative stress balance in CKD may increase the risk and extent of cardiovascular damage upon additional cardiovascular risk factors and/or events will be addressed in future studies.

Maternal binge alcohol consumption leads to distinctive acute perturbations in embryonic cardiac gene expression profiles.

Excessive alcohol consumption during pregnancy is associated with high risk of congenital heart defects, but it is unclear how alcohol specifically affects heart development during the acute aftermath of a maternal binge drinking episode. We hypothesize that administration of a single maternal binge dose of alcohol to pregnant mice at embryonic day 9.5 (E9.5) causes perturbations in the expression patterns of specific genes in the developing heart in the acute period (1-3 days) following the binge episode. To test this hypothesis and identify strong candidate ethanol-sensitive target genes of interest, we adapted a mouse binge alcohol model that is associated with a high incidence of congenital heart defects as described below. Pregnant mice were administered a single dose of alcohol (2.5g/kg in saline) or control (saline alone) via oral gavage. To evaluate the impact of maternal binge alcohol on cardiac gene expression profiles, we isolated embryonic hearts from both groups (n=5/group) at 24, 48, and 72 h post-gavage for transcriptomic analyses. RNA was extracted and evaluated using quantitative RNA-sequencing (RNA-Seq) methods. To identify a cohort of binge-altered cardiac genes, we set the threshold for change at >2.0-fold difference with adjusted p < 0.05 versus control. RNA-Seq analysis of cardiac gene expression revealed that of the 17 genes that were altered within the first 48 h post-binge, with the largest category consisting of transcription factors (Alx1, Alx4, HoxB7, HoxD8, and Runx2), followed by signaling molecules (Adamts18, Dkk2, Rtl1, and Wnt7a). Furthermore, multiple comparative and pathway analyses suggested that several of the candidate genes identified through differential RNA-Seq analysis may interact through certain common pathways. To investigate this further, we performed gene-specific qPCR analyses for three representative candidate targets: Runx2, Wnt7a, and Mlxipl. Notably, only Wnt7a showed significantly (p < 0.05) decreased expression in response to maternal binge alcohol in the qPCR assays. These findings identify Wnt7a and a short list of potential other candidate genes and pathways for further study, which could provide mechanistic insights into how maternal binge alcohol consumption produces congenital cardiac malformations.

NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca2+ Homeostasis.

During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however. We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. We found that cardiac expression of Gαo, an inhibitory G protein encoded in humans by GNAO1, is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gαo expression increased surface sarcolemmal L-type Ca2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca2+ handling in ventricular myocytes, which led to cardiac dysfunction. These findings shed light on a novel function of Gαo in the regulation of cardiac Ca2+ homeostasis and systolic function and suggest Gαo may be an effective therapeutic target for the treatment of heart failure.

Metabolic Environment Alters the Transcriptomic Landscape of the Aged Heart

Eating disorders at older age are coupled with a number of unfavorable health outcomes, including cardiovascular disease; but mechanisms underlying the process remain to be clarified. For this purpose, we studied alterations in gene expression profile of hearts from aged mice following exposure to Western diet. Specifically, we used transgenic mice expressing the haplotype-I (Hap-I: hypertensive genotype) of the human angiotensin receptor type 1 (AT1R) gene because these animals are more prone to develop metabolic syndrome-related disorders. For comparison, mice containing haplotype-II of human AT1R (Hap-II: normotensive genotype) were used. Aged mice (20-24 months of age) were maintained on a regular diet or high fat diet with 2% NaCl (Western diet) for 16 weeks. Blood pressure was monitored by telemetry, whereas cardiac anatomy and gene expression profile were established on heart specimens, using histological and RNA-seq analysis. On regular diet, old Hap-I mice presented higher (~9 mmHg) systolic blood pressure, with respect to aged-matched Hap-II animals. Following administration of Wester diet, blood pressure increased in both groups of mice, but to a larger extent in Hap-I animals (~15 mmHg), in comparison to Hap-II (~7 mmHg). With respect to Hap-II, aged Hap-I mice on regular diet tended to have larger heart weight-to-body weight ratio, increased cell size, and higher levels of fibrosis. Western Diet treatment exacerbated these differences. RNA sequencing data from cardiac tissue of Hap-I aged mice revealed that Western diet significantly altered the expression of >500 genes (p-adj. <0.05). Bioinformatics analysis, using Qiagen IPA software, identified major alterations in main canonical pathways involved in cardiac function, inflammation, and oxidative damage. Top hits in the disease and biological function category included arrhythmia, chamber enlargement, and cell death. Importantly, IRF3, IRF7, IFNG and STAT1 were among the top upstream regulators significantly affected by Western diet. Overall, these results indicate that Western diet promotes hypertension, hypertrophy, and fibrosis in the heart of aged mice. These alterations are paralleled by perturbation of cardiac transcriptional profile, including upstream gene regulators.

Old Drug, New Trick: Tilorone, a Broad-Spectrum Antiviral Drug as a Potential Anti-Fibrotic Therapeutic for the Diseased Heart.

Cardiac fibrosis is associated with most forms of cardiovascular disease. No reliable therapies targeting cardiac fibrosis are available, thus identifying novel drugs that can resolve or prevent fibrosis is needed. Tilorone, an antiviral agent, can prevent fibrosis in a mouse model of lung disease. We investigated the anti-fibrotic effects of tilorone in human cardiac fibroblasts in vitro by performing a radioisotopic assay for [3H]-proline incorporation as a proxy for collagen synthesis. Exploratory studies in human cardiac fibroblasts treated with tilorone (10 µM) showed a significant reduction in transforming growth factor-β induced collagen synthesis compared to untreated fibroblasts. To determine if this finding could be recapitulated in vivo, mice with established pathological remodelling due to four weeks of transverse aortic constriction (TAC) were administered tilorone (50 mg/kg, i.p) or saline every third day for eight weeks. Treatment with tilorone was associated with attenuation of fibrosis (assessed by Masson’s trichrome stain), a favourable cardiac gene expression profile and no further deterioration of cardiac systolic function determined by echocardiography compared to saline treated TAC mice. These data demonstrate that tilorone has anti-fibrotic actions in human cardiac fibroblasts and the adult mouse heart, and represents a potential novel therapy to treat fibrosis associated with heart failure.

Time Course of Gene Expression Profile in Renal Ischemia and Reperfusion Injury in Mice

Ischemic renal failure is an inflammatory disease that can affect various organs, including the heart. The organ responds to the stimulus and undergoes tissue remodeling that can result in cardiac hypertrophy. This study aimed to characterize the cardiac global gene expression profile in renal ischemia/reperfusion (IR) model using microarray technology. To do that, left kidney ischemia was induced in male C57BL/6 mice for 60 minutes, followed by reperfusion (IR) for 5, 8, 15, or 20 days post ischemia (dpi). Total cardiac tissue RNA was extracted and hybridized to chips with 35,000 mouse genes. The GeneChip Mouse Genome 430 2.0 Array Expression chip (Affymetrix) was used, and CEL files generated were processed with DNA-Chip-Analyzer (dCHIP) software. Subsequent analysis considered only differences among groups of at least 1.2-fold (up or down) expression changes. Analyses of the samples indicated positive modulation of 17,413 genes and 405 pathways and negative modulation of 18,287 genes and 300 pathways. A narrower analysis of genes related to inflammation, metabolism, apoptosis, oxidative stress, and channels/ion transport was performance, and it was correlated with IR injury, corroborating previous data from literature. Renal IR induced a global shift in cardiac tissue gene expression; in particular, genes related to the inflammatory system and cardiomyocyte function were changed. The in-depth study of the cell signaling in the present study could stimulate the development of new therapeutic option to ameliorate the outcome of renal-IR-induced heart damage.

Abstract 242: Multi-omics Profiling of Rat Offspring Exposed to Gestational Diabetes Reveals Cardiometabolic Disease Development With Age

Through unknown mechanisms, fetal exposure to gestational diabetes mellitus (GDM) increases the risk for cardiovascular disease development later in life. We hypothesize that fetal exposure to GDM induces alterations in cardiomyocyte metabolism and induces left ventricular (LV) dysfunction with age. GDM was induced in female rats with a high fat (45% kcal) and sucrose diet prior to mating, throughout pregnancy and lactation. Lean control females received a low fat (10% kcal) diet. Fetal rat ventricular cardiomyocytes (FRVC) were isolated from e20.5 offspring for U-13C glucose metabolic flux analysis, mitochondrial respiration and calcium handling. The cardiac transcriptome and metabolome were measured in 3-month old offspring. LV morphology and function was assessed in the offspring from e18 to 12-months of age by transthoracic ultrasound. Offspring exposed to GDM exhibited increased LV posterior wall thickness across their life course (fetal to 12-months of age; p&lt;0.05) and impaired LV filling beginning at 6-months of age (p&lt;0.05). Consistent with the development of diastolic dysfunction in vivo, alterations in calcium flux and sarcoplasmic reticulum-dependent calcium re-uptake (1.5-fold and 1.6-fold greater, respectively) were observed in FRVC isolated from GDM offspring (p&lt;0.05). When FRVC were treated with isoproterenol, U-13C glucose metabolic flux through glycolysis and the citric acid cycle was reduced in GDM offspring, compared to Lean controls. In 3-month old offspring serum metabolomics revealed reduced citric acid cycle intermediates and an altered acylcarnitine profile. These metabolic changes corresponded to alterations in gene expression patterns identified by RNASeq associated with glucose metabolism and fatty acid transport pathways (e.g. Irs2, Slc2a4, Pfkfb2, Pdk4 and Cpt1a) . Large-scale profiling revealed GDM induced alterations in the cardiac gene expression profile leading to modified serum metabolite levels in the offspring. These alterations corresponded with mitochondrial dysfunction, impaired cardiomyocyte metabolic flux and contractility, in concert with LV hypertrophy and diastolic dysfunction in the rat offspring. Our findings identify several mechanisms that link early-life GDM exposure to the development of cardiovascular disease later in life.

Matrix metalloproteinase-7 in platelet-activated macrophages accounts for cardiac remodeling in uremic mice.

Heart failure is the leading cause of mortality in patients with end-stage renal disease, and progressive cardiac remodeling is the key pathological basis of heart failure. However, the mechanism by which uremia-induced cardiac remodeling occurs is not well understood. Here, we showed that platelets were significantly activated in 5/6 nephrectomy-operated mice, and cardiac remodeling in the uremic mice was significantly improved when platelets were effectively depleted. A cardiac fibrosis-related gene expression profile revealed that Mmp7, encoding matrix metalloproteinase-7 (MMP-7), exhibited the greatest degree of downregulation in the hearts of uremic mice with platelets depleted. Using fluorescence-activated cell sorting, we discovered that MMP-7 was mainly expressed in M1 and M4 cardiac macrophages, although it was also extensively expressed in heart tissues. For the upstream therapeutic target, neutralization of platelet factor 4 (PF4) with monoclonal antibody not only significantly suppressed M4 macrophages in vivo, but also notably prevented collagen destruction in heart tissues. For the downstream therapeutic target, the pharmacological inhibition of MMP-7 with selective inhibitor failed to notably affect the platelet status, but significantly reduced heart collagen destruction in mice, a further indication that MMP-7 is a crucial downstream molecular target of platelet activation. In vitro, platelets interacted with macrophages and drove them to upregulate MMP-7 expression via free molecules, especially PF4. Taken together, the data suggest that MMP-7 is a key downstream target of platelet activation during uremia. Thus, MMP-7 is a likely and novel therapeutic target for intervention of cardiac remodeling during uremia.

Pragmatic Analysis of Dyslipidemia Involvement in Coronary Artery Disease: A Narrative Review.

BackgroundDyslipidemia is the main factor involved in the occurrence and progression of coronary artery disease.ObjectiveThe research strategy is aimed at analyzing new data on the pathophysiology of dyslipidemia involvement in coronary artery disease, the modalities of atherogenic risk estimation and therapeutic advances.MethodsScientific articles published in PubMed from January 2017 to February 2018 were searched using the terms “dyslipidemia” and “ischemic heart disease”.ResultsPCSK9 contributes to the increase in serum levels of low-density lipoprotein-cholesterol and lipoprotein (a). The inflammation is involved in the progression of hyperlipidemia and atherosclerosis. Hypercholesterolemia changes the global cardiac gene expression profile and is thus involved in the increase of oxidative stress, mitochondrial dysfunction, and apoptosis initiated by inflammation. Coronary artery calcifications may estimate the risk of coronary events. The cardio-ankle vascular index evaluates the arterial stiffness and correlates with subclinical coronary atherosclerosis. The carotid plaque score is superior to carotid intima-media thickness for risk stratification in patients with familial hypercholesterolemia and both can independently predict coronary artery disease. The lipoprotein (a) and familial hypercholesterolemia have a synergistic role in predicting the risk of early onset and severity of coronary atherosclerosis. A decrease in atherosclerotic coronary plaque progression can be achieved in patients with plasma LDL-cholesterol levels below 70 mg/dL. A highly durable RNA interference therapeutic inhibitor of PCSK9 synthesis could be a future solution.ConclusionThe prophylaxis and treatment of coronary artery disease in a dyslipidemic patient should be based on a careful assessment of cardio-vascular risk factors and individual metabolic particularities, so it may be personalized.

Abstract 547: Metabolomic and Transcriptomic Profiling of Rat Offspring Exposed to Gestational Diabetes Reveals Altered Cardiac Gene Expression and Metabolism

Gestational diabetes mellitus (GDM) is the most common complication of pregnancy. Children exposed to GDM are at an increased risk of developing cardiometabolic diseases later in life, though the mechanisms responsible are unknown. We hypothesize that fetal exposure to GDM induces alterations in cardiomyocyte metabolism and concomitant left ventricular (LV) dysfunction with age. GDM was induced in female rats with a high fat (45% kcal) and sucrose diet prior to mating, throughout pregnancy and lactation. Lean control females received a low fat (10% kcal) diet. Fetal rat ventricular cardiomyocytes (FRVC) were isolated from e20.5 offspring for U-13C glucose metabolic flux analysis and mitochondrial respiration. Serum metabolites and cardiac transcriptome profiles were measured in 3-month old offspring. LV morphology and function was assessed through the life course of the offspring (e18 to 12-months of age) by transthoracic ultrasound. Offspring exposed to GDM exhibited increased LV posterior wall thickness across their life course (fetal to 12-months of age; p&lt;0.05) and impaired LV filling beginning at 6-months of age (p&lt;0.05). U-13C glucose metabolic flux through glycolysis and the citric acid cycle was reduced in FRVC from GDM offspring when treated with isoproterenol, and compared to Lean FRVC. Basal and maximal mitochondrial oxygen consumption was reduced for glucose (35% and 68%) and fatty acid (49% and 52%) substrates in FRVC isolated from GDM offspring (p&lt;0.05). In 3-month old GDM offspring, serum metabolomics revealed elevated levels of beta-hydroxybutyrate (2.4-fold, p&lt;0.05) and reduced levels of several citric acid cycle intermediates, which also corresponded to alterations in gene expression patterns identified by RNASeq transcript analysis. Large-scale profiling revealed GDM induced alterations in the cardiac gene expression profile leading to modified serum metabolite levels in the offspring. These alterations corresponded with mitochondrial dysfunction, impaired cardiomyocyte metabolic flux and contractility, in concert with LV hypertrophy and diastolic dysfunction in the rat offspring. Our findings identify several mechanisms that link early-life GDM exposure to the development of cardiovascular disease later in life.