Changes In Cardiac Gene Expression Research Articles

Elimination of CaMKIIδ Autophosphorylation by CRISPR-Cas9 Base Editing Improves Survival and Cardiac Function in Heart Failure in Mice.

Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic β-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.

Maternal use of methamphetamine alters cardiovascular function in the adult offspring.

Methamphetamine is one of the most commonly used illicit drugs during pregnancy. Most studies investigating the impact of maternal use of methamphetamine on children have focused on neurological outcomes. In contrast, cardiovascular outcomes in these children have not been characterized. Recent studies in rodents provide evidence that prenatal exposure to methamphetamine induces changes in cardiac gene expression, changes in the heart's susceptibility to ischemic injury, and changes in vascular function that may increase the risk of developing cardiovascular disorders later in life. Importantly, these changes are sex-dependent. This review summarizes our current understanding of how methamphetamine use during pregnancy impacts the cardiovascular function of adult offspring and highlights gaps in our knowledge of the potential cardiovascular risks associated with prenatal exposure to methamphetamine.

Maternal use of methamphetamine induces sex-dependent changes in myocardial gene expression in adult offspring.

Methamphetamine is a commonly abused illicit stimulant that has prevalent use among women of child-bearing age. While there are extensive studies on the neurological effects of prenatal methamphetamine exposure, relatively little is known about the effect of prenatal methamphetamine on the adult cardiovascular system. Earlier work demonstrated that prenatal methamphetamine exposure sex dependently (females only) sensitizes the adult heart to ischemic injury. These data suggest that prenatal exposure to methamphetamine may induce sex-dependent changes in cardiac gene expression that persist in adult offspring. The goal of this study was to test the hypothesis that prenatal methamphetamine exposure induces changes in cardiac gene expression that persist in the adult heart. Hearts of prenatally exposed female offspring exhibited a greater number of changes in gene expression compared to male offspring (184 changes compared with 74 in male offspring and 89 changes common between both sexes). Dimethylarginine dimethylaminohydrolase 2 and 3-hydroxybutyrate dehydrogenase 1 (genes implicated in heart failure) were shown by Western Blot to be under expressed in adult females that were prenatally exposed to methamphetamine, while males were deficient in 3-Hydroxybutyrate Dehydrogenase 1 only. These data indicate that prenatal methamphetamine exposure induces changes in gene expression that persist into adulthood. This is consistent with previous findings that prenatal methamphetamine sex dependently sensitizes the adult heart to ischemic injury and may increase the risk of developing cardiac disorders during adulthood.

Transcriptomic analysis of cardiac gene expression across the life course in male and female mice.

Risk for heart disease increases with advanced age and differs between sexes, with females generally protected from heart disease until menopause. Despite these epidemiological observations, the molecular mechanisms that underlie sex‐specific differences in cardiac function have not been fully described. We used high throughput transcriptomics in juvenile (5 weeks), adult (4–6 months), and aged (18 months) male and female mice to understand how cardiac gene expression changes across the life course and by sex. While male gene expression profiles differed between juvenile‐adult and juvenile‐aged (254 and 518 genes, respectively), we found no significant differences in adult‐aged gene expression. Females had distinct gene expression changes across the life course with 1835 genes in juvenile‐adult and 1328 in adult‐aged. Analysis of differentially expressed genes (DEGs) suggests that juvenile to adulthood genes were clustered in cell cycle and development‐related pathways in contrast to adulthood‐aged which were characterized by immune‐and inflammation‐related pathways. Analysis of sex differences within each age suggests that juvenile and aged cardiac transcriptomes are different between males and females, with significantly fewer DEGs identified in adult males and females. Interestingly, the male–female differences in early age were distinct from those in advanced age. These findings are in contrast to expected sex differences historically attributed to estrogen and could not be explained by estrogen‐direct mechanisms alone as evidenced by juvenile sexual immaturity and reproductive incompetence in the aged mice. Together, distinct trajectories in cardiac transcriptomic profiles highlight fundamental sex differences across the life course and demonstrate the need for the consideration of age and sex as biological variables in heart disease.

Transcriptomic Analysis Of Cardiac Gene Expression Across The Life‐Course In Male And Female Mice

Risk for heart disease increases with advanced age and differs between sexes, with females generally protected from heart disease until menopause. Despite these epidemiological and clinical observations, however, the molecular mechanisms of the cardiac aging life-course and how it differs between sexes is not fully described. We used high throughput transcriptomics in juvenile (5 weeks), adult (4-6 months), and aged (18 months) to understand how cardiac gene expression changes across the life-course and by sex. Age-related changes in cardiac gene expression differed significantly between males and females. While male gene expression changed from the juvenile-adult and juvenile-aged period (203 and 395 genes, respectively), adult-old gene expression was unchanged. Female gene expression changed across the life-course with 2009 genes from juvenile-adult and 1656 from adult-aged. Gene enrichment analysis of differentially expressed genes (DEG) suggests that juvenile to adulthood genes are clustered in cell-cycle and development-related pathways in contrast to adulthood to aged shift with immune and inflammation-related pathways. Within age, significant sex differences existed as well. Juvenile and aged male and female mice displayed significantly different gene expression, while adult male and female gene expression was similar. Interestingly, there was very little overlap in differently expressed genes juvenile and aged mice, suggesting sex differences in early age are distinct from those in advanced age. Gene enrichment analysis shows early juvenile DEGs clustering in cell-differentiation and development pathways, aged mice DEGs clustering in protein metabolism and its regulation, and suppression of some immune response pathways. These findings are contrary to expected sex differences historically attributed to estrogen, given that juvenile female mice are not yet sexually mature and the aged female cohort demonstrates cessation of estrous cycle. Thus, we suggest that mechanisms beyond sex hormones likely explain these significant sex-specific differences across the mouse life-course. Together, distinct trajectories in transcriptomic profile hints to fundamental age-sex-differences in cardiac aging and demonstrates the need for consideration of age and sex as biological variables in heart disease.

The long‐noncoding RNA from the MYH7b locus (lncMYH7b) has pronounced effects on cardiomyocyte molecular and cellular phenotypes

Myosin heavy chain 7b (MYH7b)is annotated as an ancient gene that encodes a myosin II motor. A conserved exon-skipping mechanism prevents the expression of cardiac protein; however a long-noncoding RNA (lncMYH7b) and miRNA (miR-499) are expressed. While the roles of miR-499 in the heart are well-defined, it has been hypothesized that lncMYH7b RNA transcription is maintained to preserve miR-499 expression. Surprisingly, we have found that lncMYH7b plays an additional role in the cardiac transcriptome. In fact, lncMYH7b RNA knockdown in human iPS-derived cardiomyocytes triggers several changes in cardiac gene expression and function As an example, knockdown cells exhibited a slower beat rate and a significant reduction in sarcomere number. Potentially contributing to these phenotypes are decreased expression of adrenergic signaling proteins and the loss of formin homology 2 domain containing 3 protein (FHOD3), respectively. Perhaps most striking, as a result of knocking down lncMYH7b RNA, the important ratio of β- and α-myosin heavy chains was decreased at both the RNA and protein level. This is likely due to the loss of expression of TEAD3, a transcription factor known to target β-myosin in cardiomyocytes. Our data show that lncMYH7b RNA is a novel example of an ancient gene fulfilling different roles in the cell based on its alternative splicing. Furthermore, a shift in the myosin ratio is a molecular hallmark of heart failure, positioning this pathway as a therapeutic target for therapeutic intervention.

Mineralocorticoid receptor antagonism by finerenone is sufficient to improve function in preclinical muscular dystrophy.

AimsDuchenne muscular dystrophy (DMD) is an X‐linked inherited disease due to dystrophin deficiency causing skeletal and cardiac muscle dysfunction. Affected patients lose ambulation by age 12 and usually die in the second to third decades of life from cardiac and respiratory failure. Symptomatic treatment includes the use of anti‐inflammatory corticosteroids, which are associated with side effects including weight gain, osteoporosis, and increased risk of cardiovascular disease. Novel treatment options include blockade of the renin–angiotensin–aldosterone system, because angiotensin as well as aldosterone contribute to persistent inflammation and fibrosis, and aldosterone blockade represents an efficacious anti‐fibrotic approach in cardiac failure. Recent preclinical findings enabled successful clinical testing of a combination of steroidal mineralocorticoid receptor antagonists (MRAs) and angiotensin converting enzyme inhibitors in DMD boys. The efficacy of MRAs alone on dystrophic skeletal muscle and heart has not been investigated. Here, we tested efficacy of the novel non‐steroidal MRA finerenone as a monotherapy in a preclinical DMD model.Methods and resultsThe dystrophin‐deficient, utrophin haploinsufficient mouse model of DMD was treated with finerenone and compared with untreated dystrophic and wild‐type controls. Grip strength, electrocardiography, cardiac magnetic resonance imaging, muscle force measurements, histological quantification, and gene expression studies were performed. Finerenone treatment alone resulted in significant improvements in clinically relevant functional parameters in both skeletal muscle and heart. Normalized grip strength in rested dystrophic mice treated with finerenone (40.3 ± 1.0 mN/g) was significantly higher (P = 0.0182) compared with untreated dystrophic mice (35.2 ± 1.5 mN/g). Fatigued finerenone‐treated dystrophic mice showed an even greater relative improvement (P = 0.0003) in normalized grip strength (37.5 ± 1.1 mN/g) compared with untreated mice (29.7 ± 1.1 mN/g). Finerenone treatment also led to significantly lower (P = 0.0075) susceptibility to limb muscle damage characteristic of DMD measured during a contraction‐induced injury protocol. Normalized limb muscle force after five lengthening contractions resulted in retention of 71 ± 7% of baseline force in finerenone‐treated compared with only 51 ± 4% in untreated dystrophic mice. Finerenone treatment also prevented significant reductions in myocardial strain rate (P = 0.0409), the earliest sign of DMD cardiomyopathy. Moreover, treatment with finerenone led to very specific cardiac gene expression changes in clock genes that might modify cardiac pathophysiology in this DMD model.ConclusionsFinerenone administered as a monotherapy is disease modifying for both skeletal muscle and heart in a preclinical DMD model. These findings support further evaluation of finerenone in DMD clinical trials.

Cardiac Gene Expression and Histology in a Rat Model of Fat Embolism

IntroductionFat Embolism consequent to Triolein (T) intravenous injection in rats induces severe histopathologic pulmonary damage at 48 hours mostly related to vasculitis, peribronchial and septal inflammation with an increase in mast cells, and eventually, organ fibrosis. We previously reported that the hearts of the same animals at 48 hours did not show vasculitis of coronary arteries branches, inflammation or an increase in mast cells. The objective of this study was to determine if T treatment by 10 weeks induced changes in cardiac gene expression or histopathology related to fat embolism.Materials and Methods24 Sprague Dawley Rats (250–330g) were divided into two groups, 48 hour treatment and 10 week treatment (N=12 each). Six rats of each group were intravenously injected with 0.2 ml of T, and the other 6, 0.2 ml of normal saline. The rats were euthanized under general anesthesia 48 hours or 10 weeks after injection. A cut was made in hearts along the transverse plane; the top half of hearts were snap frozen in liquid nitrogen for gene expression studies and the bottom half was fixed in formaldehyde for histopathological evaluation similar to lung preparations. After RNA isolation, real time RT‐PCR was performed on genes associated with pathological cardiac hypertrophy or fibrosis (beta myosin heavy chain, alpha myosin heavy chain, skeletal muscle alpha actin, atrial natriuretic peptide, brain natriuretic peptide, vimentin, and collagen 1a1).ResultsDespite significant damage to the lungs, there was no significant changes in expression of any of these genes in the hearts of the rats treated with T, versus those of the saline treated group, whether sacrificed at 48 hours or 10 weeks post injection (P>0.05 ANOVA). Histopathological evaluation of the hearts at 10 weeks also did not show any damage in the myocardium in sections stained by H and E and no vasculitis, mast cell increase, or ventricular enlargements. Trichrome staining did not show any fibrotic increase in hearts of T injected animals, thus confirming the previous observations and gene expression analysis.ConclusionThere was a marked contrast in organ damage between hearts and lungs in this study in rats with T treatment. Hearts demonstrated no significant change in gene expression or histology demonstrating a lack of fibroblast infiltration, hypertrophy, or remodeling due to T treatment. Kidney, liver, brains, and aortas also showed varying degrees of histopathological damage compared to the severity of the damage of the lungs. The renin angiotensin system plays a key role in the pathogenesis of the lung damage with fat embolism and this system needs to be further explored for organ differences to fat embolism induced by T treatment.Support or Funding InformationSupported by the Catherine T. Geldmacher Foundation, St. Louis, MO

Abstract 945: A Novel Role of Cardiac DNA Methylation as a Regulator of Fibrosis in Human Diabetic Heart Failure

Human heart failure (HF) is accompanied by changes in cardiac gene expression; however, the impact of diabetes mellitus on this transcriptional regulation remains unclear. Our laboratory studies the role of epigenetics in HF and focuses on DNA methylation, commonly accepted as a negative regulator of gene expression when present in a gene’s promoter. In the current study, we hypothesized that diabetes alters cardiac DNA methylation and the transcriptome. We performed an integrated analysis of RNA-sequencing and DNA methylation using human left ventricle biopsies from HF patients to determine the effect of diabetes on this reprogramming. Our analysis identified global changes in cardiac DNA methylation as well as mRNA expression sufficient to distinguish diabetic from non-diabetic patients. Specifically, we identified significant regulation of 1,133 genes (|1.5| fold-change, P<0.05) and 9,302 methylation sites (|5%| methylation, P<0.05). Within the 391 co-regulated genes, only 174 genes had altered promoter-associated methylation. Consistent with the accepted dogma that promoter methylation inversely regulates gene expression, we found that 119 of those 174 genes displayed this inverse relationship; of these, an overwhelming majority (102) were hypomethylated in diabetic relative to non-diabetic hearts. These findings contrast the cardiac hypermethylation we have reported for ischemic HF. Gene set enrichment of the 119 inversely-regulated genes identified numerous pathways involved in fibrosis, including extracellular matrix organization (FDR<0.05, 3.9% enriched) and collagen biosynthesis (FDR<0.05, 6.3% enriched), which suggest that DNA methylation contributes to the adverse cardiac remodeling associated with diabetic heart failure. To identify putative regulators of these transcriptional and epigenetic differences, a candidate gene approach was used to reveal induction (2.5-fold, Q<0.05) and demethylation (20.5%, P<0.05) of EGR2, a known regulator of fibrosis. Furthermore, we found induction of GADD45beta (2.6-fold, Q < 0.05) a known regulator of DNA demethylation. Taken together, these observations suggest that epigenetic mechanisms underlie an etiology-specific reprogramming of cardiac fibrosis in diabetic HF.

Deletion of the Cardiomyocyte Glucocorticoid Receptor Leads to Sexually Dimorphic Changes in Cardiac Gene Expression and Progression to Heart Failure.

BackgroundThe contribution of glucocorticoids to sexual dimorphism in the heart is essentially unknown. Therefore, we sought to determine the sexually dimorphic actions of glucocorticoid signaling in cardiac function and gene expression. To accomplish this goal, we conducted studies on mice lacking glucocorticoid receptors (GR) in cardiomyocytes (cardioGRKO mouse model).Methods and ResultsDeletion of cardiomyocyte GR leads to an increase in mortality because of the development of spontaneous cardiac pathology in both male and female mice; however, females are more resistant to GR signaling inactivation in the heart. Male cardioGRKO mice had a median survival age of 6 months. In contrast, females had a median survival age of 10 months. Transthoracic echocardiography data showed phenotypic differences between male and female cardioGRKO hearts. By 3 months of age, male cardioGRKO mice exhibited left ventricular systolic dysfunction. Conversely, no significant functional deficits were observed in female cardioGRKO mice at the same time point. Functional sensitivity of male hearts to the loss of cardiomyocyte GR was reversed following gonadectomy. RNA‐Seq analysis showed that deleting GR in the male hearts leads to a more profound dysregulation in the expression of genes implicated in heart rate regulation (calcium handling). In agreement with these gene expression data, cardiomyocytes isolated from male cardioGRKO hearts displayed altered intracellular calcium responses. In contrast, female GR‐deficient cardiomyocytes presented a response comparable with controls.ConclusionsThese data suggest that GR regulates calcium responses in a sex‐biased manner, leading to sexually distinct responses to stress in male and female mice hearts, which may contribute to sex differences in heart disease, including the development of ventricular arrhythmias that contribute to heart failure and sudden death.

Endothelial to Mesenchymal Transition in the Rat Heart Following Exposure to a Model of Posttraumatic Stress Disorder

People who are exposed to life‐threatening trauma are at risk of developing posttraumatic stress disorder (PTSD). In addition to psychological manifestations, PTSD is associated with increased risk of myocardial infarction and arrhythmias. We previously reported that rats exposed to a predator‐based model of PTSD develop myocardial hypersensitivity to ischemic injury. The present work provided mechanistic insight into changes in cardiac gene expression and histology that may predispose the heart to injury induced by an ischemic insult. Male rats were subjected to two cat exposures (separated by a period of 10 days) and daily cage‐mate changes for 31 days. Control rats were not exposed to the cat or cage‐mate changes. Ventricular tissue was analyzed by RNA sequencing, western blotting, histology, and immunohistochemistry. Gene expression analysis identified clusters of upregulated genes associated with endothelial to mesenchymal transition in hearts from stressed rats. This was supported by upregulation of α‐smooth muscle actin and a decrease in the number of CD31 positive endothelial cells. Multifocal lesions characterized by necrosis, mononuclear cell infiltration, and collagen deposition were observed in hearts from all stressed rats but not in any of the control rats. These changes in gene expression and phenotype are indicative of endothelial to mesenchymal transition and may underlie the myocardial hypersensitivity to ischemic injury that develops in these animals. Similar changes in the human heart may also underlie the increased frequency of myocardial infarction, arrhythmias, and other cardiac abnormalities in humans with PTSD.Support or Funding InformationThis work was supported by grant R15HL132322 awarded to BRR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NBCe1 Na+-HCO3- cotransporter ablation causes reduced apoptosis following cardiac ischemia-reperfusion injury in vivo.

AIMTo investigate the hypothesis that cardiomyocyte-specific loss of the electrogenic NBCe1 Na+-HCO3- cotransporter is cardioprotective during in vivo ischemia-reperfusion (IR) injury.METHODSAn NBCe1 (Slc4a4 gene) conditional knockout mouse (KO) model was prepared by gene targeting. Cardiovascular performance of wildtype (WT) and cardiac-specific NBCe1 KO mice was analyzed by intraventricular pressure measurements, and changes in cardiac gene expression were determined by RNA Seq analysis. Response to in vivo IR injury was analyzed after 30 min occlusion of the left anterior descending artery followed by 3 h of reperfusion.RESULTSLoss of NBCe1 in cardiac myocytes did not impair cardiac contractility or relaxation under basal conditions or in response to β-adrenergic stimulation, and caused only limited changes in gene expression patterns, such as those for electrical excitability. However, following ischemia and reperfusion, KO heart sections exhibited significantly fewer apoptotic nuclei than WT sections.CONCLUSIONThese studies indicate that cardiac-specific loss of NBCe1 does not impair cardiovascular performance, causes only minimal changes in gene expression patterns, and protects against IR injury in vivo .

Ectopic expression of Cdk8 induces eccentric hypertrophy and heart failure.

Widespread changes in cardiac gene expression occur during heart failure, yet the mechanisms responsible for coordinating these changes remain poorly understood. The Mediator complex represents a nodal point for modulating transcription by bridging chromatin-bound transcription factors with RNA polymerase II activity; it is reversibly regulated by its cyclin-dependent kinase 8 (Cdk8) kinase submodule. Here, we identified increased Cdk8 protein expression in human failing heart explants and determined the consequence of this increase in cardiac-specific Cdk8-expressing mice. Transgenic Cdk8 overexpression resulted in progressive dilated cardiomyopathy, heart failure, and premature lethality. Prior to functional decline, left ventricular cardiomyocytes were dramatically elongated, with disorganized transverse tubules and dysfunctional calcium handling. RNA sequencing results showed that myofilament gene isoforms not typically expressed in adult cardiomyocytes were enriched, while oxidative phosphorylation and fatty acid biosynthesis genes were downregulated. Interestingly, candidate upstream transcription factor expression levels and MAPK signaling pathways thought to determine cardiomyocyte size remained relatively unaffected, suggesting that Cdk8 functions within a novel growth regulatory pathway. Our findings show that manipulating cardiac gene expression through increased Cdk8 levels is detrimental to the heart by establishing a transcriptional program that induces pathological remodeling and eccentric hypertrophy culminating in heart failure.

The von Hippel-Lindau Chuvash mutation in mice alters cardiac substrate and high-energy phosphate metabolism.

Hypoxia-inducible factor (HIF) appears to function as a global master regulator of cellular and systemic responses to hypoxia. HIF pathway manipulation is of therapeutic interest; however, global systemic upregulation of HIF may have as yet unknown effects on multiple processes. We used a mouse model of Chuvash polycythemia (CP), a rare genetic disorder that modestly increases expression of HIF target genes in normoxia, to understand what these effects might be within the heart. An integrated in and ex vivo approach was employed. Compared with wild-type controls, CP mice had evidence (using in vivo magnetic resonance imaging) of pulmonary hypertension, right ventricular hypertrophy, and increased left ventricular ejection fraction. Glycolytic flux (measured using [(3)H]glucose) in the isolated contracting perfused CP heart was 1.8-fold higher. Net lactate efflux was 1.5-fold higher. Furthermore, in vivo (13)C-magnetic resonance spectroscopy (MRS) of hyperpolarized [(13)C1]pyruvate revealed a twofold increase in real-time flux through lactate dehydrogenase in the CP hearts and a 1.6-fold increase through pyruvate dehydrogenase. (31)P-MRS of perfused CP hearts under increased workload (isoproterenol infusion) demonstrated increased depletion of phosphocreatine relative to ATP. Intriguingly, no changes in cardiac gene expression were detected. In summary, a modest systemic dysregulation of the HIF pathway resulted in clear alterations in cardiac metabolism and energetics. However, in contrast to studies generating high HIF levels within the heart, the CP mice showed neither the predicted changes in gene expression nor any degree of LV impairment. We conclude that the effects of manipulating HIF on the heart are dose dependent.

Ecstasy (MDMA) Alters Cardiac Gene Expression and DNA Methylation: Implications for Circadian Rhythm Dysfunction in the Heart.

MDMA (ecstasy) is an illicit drug that stimulates monoamine neurotransmitter release and inhibits reuptake. MDMA's acute cardiotoxicity includes tachycardia and arrhythmia which are associated with cardiomyopathy. MDMA acute cardiotoxicity has been explored, but neither long-term MDMA cardiac pathological changes nor epigenetic changes have been evaluated. Microarray analyses were employed to identify cardiac gene expression changes and epigenetic DNA methylation changes. To identify permanent MDMA-induced pathogenetic changes, mice received daily 10- or 35-day MDMA, or daily 10-day MDMA followed by 25-day saline washout (10 + 25 days). MDMA treatment caused differential gene expression (p < .05, fold change >1.5) in 752 genes following 10 days, 558 genes following 35 days, and 113 genes following 10-day MDMA + 25-day saline washout. Changes in MAPK and circadian rhythm gene expression were identified as early as 10 days. After 35 days, circadian rhythm genes (Per3, CLOCK, ARNTL, and NPAS2) persisted to be differentially expressed. MDMA caused DNA hypermethylation and hypomethylation that was independent of gene expression; hypermethylation of genes was found to be 71% at 10 days, 68% at 35 days, and 91% at 10 + 25 days washout. Differential gene expression paralleled DNA methylation in 22% of genes at 10-day treatment, 17% at 35 days, and 48% at 10 + 25 days washout. We show here that MDMA induced cardiac epigenetic changes in DNA methylation where hypermethylation predominated. Moreover, MDMA induced gene expression of key elements of circadian rhythm regulatory genes. This suggests a fundamental organism-level event to explain some of the etiologies of MDMA dysfunction in the heart.

The effect of a preparation of minerals, vitamins and trace elements on the cardiac gene expression pattern in male diabetic rats

BackgroundDiabetic patients have an increased risk of developing cardiovascular diseases, which are the leading cause of death in developed countries. Although multivitamin products are widely used as dietary supplements, the effects of these products have not been investigated in the diabetic heart yet. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) affects the cardiac gene expression pattern in experimental diabetes.MethodsTwo-day old male Wistar rats were injected with streptozotocin (i.p. 100 mg/kg) or citrate buffer to induce diabetes. From weeks 4 to 12, rats were fed with a vehicle or a MVT preparation. Fasting blood glucose measurement and oral glucose tolerance test were performed at week 12, and then total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41012 oligonucleotides.ResultsSignificantly elevated fasting blood glucose concentration and impaired glucose tolerance were markedly improved by MVT-treatment in diabetic rats at week 12. Genes with significantly altered expression due to diabetes include functional clusters related to cardiac hypertrophy (e.g. caspase recruitment domain family, member 9; cytochrome P450, family 26, subfamily B, polypeptide; FXYD domain containing ion transport regulator 3), stress response (e.g. metallothionein 1a; metallothionein 2a; interleukin-6 receptor; heme oxygenase (decycling) 1; and glutathione S-transferase, theta 3), and hormones associated with insulin resistance (e.g. resistin; FK506 binding protein 5; galanin/GMAP prepropeptide). Moreover the expression of some other genes with no definite cardiac function was also changed such as e.g. similar to apolipoprotein L2; brain expressed X-linked 1; prostaglandin b2 synthase (brain). MVT-treatment in diabetic rats showed opposite gene expression changes in the cases of 19 genes associated with diabetic cardiomyopathy. In healthy hearts, MVT-treatment resulted in cardiac gene expression changes mostly related to immune response (e.g. complement factor B; complement component 4a; interferon regulatory factor 7; hepcidin).ConclusionsMVT-treatment improved diagnostic markers of diabetes. This is the first demonstration that MVT-treatment significantly alters cardiac gene expression profile in both control and diabetic rats. Our results and further studies exploring the mechanistic role of individual genes may contribute to the prevention or diagnosis of cardiac complications in diabetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0248-6) contains supplementary material, which is available to authorized users.

Abstract 11988: Next Generation Sequencing of Cardiac Gene Expression Changes Associated With Heart Failure in Chronic Inflammation Inhibition

Since a variety of gene alternations are involved in heart failure (HF), identifying key functional molecules associated with HF is a challenging task. Therefore, we investigated the global transcriptome analysis of left ventricle (LV) from tachycardia induced HF dogs using next generation genome sequencers. The mRNA sequence reads aligned with ~15000 of the 21407 genes using the human genome19 database. The analysis identified significantly up-regulated genes associated with inflammation such as Rel A, STAT 3, RIPK2, CDKN1, TNF-alpha, interleukin (IL) and p53 related pathways and collagen type 4, which regulate cell cycles of inflammatory cells and fibroblast. These data suggest chronic inflammation induced by those novel genes may affect cardiac remodelling and inhibition of these effects may become one of the strategies to treat HF. Thus, we focused on cardiac chymase activity that is stored in the mast cells recruited by stem cell factor and affects chronic inflammation closely linked with cardiac remodelling. We examined therapeutic effects of a chymase inhibitor (ChyI 0.3mg/kg/day, n=7) on cardiac structural changes and gene expressions in failing dogs’ heart. In HF, the number of chymase-degranulated cells evaluated by chymotrypsin staining increased in the LV compared with the normal group, however, ChyI significantly decreased that cell density. ChyI significantly suppressed the IL-6, Rel A, STAT 3, CDKN1, RIPK2, TNF-alpha and p-53 mRNA levels in the cardiac tissue. Not only collagen type 1,3 but also type 4 mRNA involved in maintaining the structural integrity were decreased compared with the vehicle. ChyI suppressed fibrosis in LV evaluated with picrosirius red stain (% collagen volume fraction; 1.1, Normal; 4.6, ChyI; vs 7.1%, Vehicle; P&lt;0.05, respectively). Despite no significant differences in blood pressure, ChyI shortened the prolongation of tau (22, Normal; 35, ChyI; vs 50 msec, Vehicle; P&lt;0.05, respectively) and decreased LV end-diastolic pressure. We can conclude that the global transcriptome analysis can provide new insights into cellular mechanisms and therapeutic effects of ChyI in HF.

Caffeine induces both short‐term and long‐term effects on gene expression and DNA methylation in the mouse heart (542.3)

Introduction: Evidence indicates that disruption of normal prenatal development influences an individual’s risk of developing cardiovascular disease as adults. Thus, understanding how in utero exposure to chemical agents leads to increased susceptibility to adult diseases is a critical health related issue. One substance that fetuses are commonly exposed is caffeine, an adenosine receptor antagonist, with consumption during the first month of pregnancy being reported by 60% of women. Methods: Pregnant CD‐1 dams were injected with 20 mg/kg of caffeine once a day from E6.5‐E9.5. Hypothesis: Altered adenosine action in utero leads to adverse effects on adult heart function and morphology, and these effects are mediated by DNA methylation‐induced changes in cardiac gene expression. Results: Using an outbred strain of mouse, CD‐1, we demonstrated that in utero caffeine exposure induces increased wall thickness and increased left ventricular mass in adult hearts. When we examined gene expression in embryonic hearts following the same caffeine treatment, we demonstrated that enzymes responsible for DNA methylation and demethylation were affected. The DNA methylation genes DNA methyltransferase 1 and 3a were both down regulated and the Tet 1 enzyme which initiates DNA demethylation was up regulated following in utero caffeine exposure. These results are consistent with our observations that in utero caffeine exposure results in global hypomethylation in the adult heart. In addition, the cardiac structural genes Myh7, titan, and troponin I1 had altered gene expression. Conclusion: Caffeine affects the expression level of genes that control DNA methylation in utero, and caffeine leads to altered gene expression and cardiac morphology in adulthood.Grant Funding Source: R01 HD058086

Integrated mechanisms of CaMKII-dependent ventricular remodeling

CaMKII has been shown to be activated during different cardiac pathological processes, and CaMKII-dependent mechanisms contribute to pathological cardiac remodeling, cardiac arrhythmias, and contractile dysfunction during heart failure. Activation of CaMKII during cardiac stress results in a broad number of biological effects such as, on the one hand, acute effects due to phosphorylation of distinct cellular proteins as ion channels and calcium handling proteins and, on the other hand, integrative mechanisms by changing gene expression. This review focuses on transcriptional and epigenetic effects of CaMKII activation during chronic cardiac remodeling. Multiple mechanisms have been described how CaMKII mediates changes in cardiac gene expression. CaMKII has been shown to directly phosphorylate components of the cardiac gene regulation machinery. CaMKII phosphorylates several transcription factors such as CREB that induces the activation of specific gene programs. CaMKII activates transcriptional regulators also indirectly by phosphorylating histone deacetylases, especially HDAC4, which in turn inhibits transcription factors that drive cardiac hypertrophy, fibrosis, and dysfunction. Recent studies demonstrate that CaMKII also phosphorylate directly histones, which may contribute to changes in gene expression. These findings of CaMKII-dependent gene regulation during cardiac remodeling processes suggest novel strategies for CaMKII-dependent “transcriptional or epigenetic therapies” to control cardiac gene expression and function. Manipulation of CaMKII-dependent signaling pathways in the settings of pathological cardiac growth, remodeling, and heart failure represents an auspicious therapeutic approach.

Targeted Ablation of Nesprin 1 and Nesprin 2 from Murine Myocardium Results in Cardiomyopathy, Altered Nuclear Morphology and Inhibition of the Biomechanical Gene Response

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.