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1.  Partial Deletion of eNOS Gene Causes Hyperinsulinemic State, Unbalance of Cardiac Insulin Signaling Pathways and Coronary Dysfunction Independently of High Fat Diet 
PLoS ONE  2014;9(8):e104156.
Abnormalities in eNOS gene, possibly interacting with high fat diet (HFD), affect peripheral vascular function and glucose metabolism. The relative role of eNOS gene, HFD and metabolic derangement on coronary function has not been fully elucidated. We test whether eNOS gene deficiency per se or in association with HFD modulates coronary function through mechanisms involving molecular pathways related to insulin signaling. Wild type (WT), eNOS−/− and eNOS+/− mice were studied. WT and eNOS+/− mice were fed with either standard or HF diet for 16 weeks and compared with standard diet fed eNOS−/−. Glucose and insulin tolerance tests were performed during the last week of diet. Coronary resistance (CR) was measured at baseline and during infusions of acetylcholine (Ach) or sodium-nitroprusside (SNP) to evaluate endothelium-dependent or independent vasodilation, in the Langendorff isolated hearts. Cardiac expression of Akt and ERK genes as evaluation of two major insulin-regulated signaling pathways involved in the control of vascular tone were assessed by western blot. HFD-fed mice developed an overt diabetic state. Conversely, chow-fed genetically modified mice (in particular eNOS−/−) showed a metabolic pattern characterized by normoglycemia and hyperinsulinemia with a limited degree of insulin resistance. CR was significantly higher in animals with eNOS gene deletions than in WT, independently of diet. Percent decrease in CR, during Ach infusion, was significantly lower in both eNOS−/− and eNOS+/− mice than in WT, independently of diet. SNP reduced CR in all groups except eNOS−/−. The cardiac ERK1-2/Akt ratio, increased in animals with eNOS gene deletions compared with WT, independently of diet. These results suggest that the eNOS genetic deficiency, associated or not with HFD, has a relevant effect on coronary vascular function, possibly mediated by increase in blood insulin levels and unbalance in insulin-dependent signaling in coronary vessels, consistent with a shift towards a vasoconstrictive pattern.
doi:10.1371/journal.pone.0104156
PMCID: PMC4122412  PMID: 25093405
2.  Sequencing of NOTCH1, GATA5, TGFBR1 and TGFBR2 genes in familial cases of bicuspid aortic valve 
BMC Medical Genetics  2013;14:44.
Background
The purpose of our study was to investigate the potential contribution of germline mutations in NOTCH1, GATA5 and TGFBR1 and TGFBR2 genes in a cohort of Italian patients with familial Bicuspid Aortic Valve (BAV).
Methods
All the coding exons including adjacent intronic as well as 5′ and 3′ untranslated (UTR) sequences of NOTCH1, GATA5, TGFBR1 and TGFBR2 genes were screened by direct gene sequencing in 11 index patients (8 males; age = 42 ± 19 years) with familial BAV defined as two or more affected members.
Results
Two novel mutations, a missense and a nonsense mutation (Exon 5, p.P284L; Exon 26, p.Y1619X), were found in the NOTCH1 gene in two unrelated families. The mutations segregated with the disease in these families, and they were not found on 200 unrelated chromosomes from ethnically matched controls. No pathogenetic mutation was identified in GATA5, TGFBR1 and TGFBR2 genes.
Conclusions
Two novel NOTCH1 mutations were identified in two Italian families with BAV, highlighting the role of a NOTCH1 signaling pathway in BAV and its aortic complications. These findings are of relevance for genetic counseling and clinical care of families presenting with BAV. Future studies are needed in order to unravel the still largely unknown genetics of BAV.
doi:10.1186/1471-2350-14-44
PMCID: PMC3637327  PMID: 23578328
Bicuspid aortic valve; Direct gene sequencing; Genes
3.  Next generation sequencing in cardiovascular diseases 
World Journal of Cardiology  2012;4(10):288-295.
In the last few years, the advent of next generation sequencing (NGS) has revolutionized the approach to genetic studies, making whole-genome sequencing a possible way of obtaining global genomic information. NGS has very recently been shown to be successful in identifying novel causative mutations of rare or common Mendelian disorders. At the present time, it is expected that NGS will be increasingly important in the study of inherited and complex cardiovascular diseases (CVDs). However, the NGS approach to the genetics of CVDs represents a territory which has not been widely investigated. The identification of rare and frequent genetic variants can be very important in clinical practice to detect pathogenic mutations or to establish a profile of risk for the development of pathology. The purpose of this paper is to discuss the recent application of NGS in the study of several CVDs such as inherited cardiomyopathies, channelopathies, coronary artery disease and aortic aneurysm. We also discuss the future utility and challenges related to NGS in studying the genetic basis of CVDs in order to improve diagnosis, prevention, and treatment.
doi:10.4330/wjc.v4.i10.288
PMCID: PMC3482622  PMID: 23110245
Next generation sequencing; Genetics of cardiovascular diseases; Cardiomyopathies; Coronary artery disease; Complex disease
4.  T−786→C polymorphism of the endothelial nitric oxide synthase gene is associated with insulin resistance in patients with ischemic or non ischemic cardiomyopathy 
BMC Medical Genetics  2012;13:92.
Background
Insulin resistance (IR) and endothelial dysfunction are frequently associated in cardiac disease. The T−786→C variant in the promoter region of the endothelial nitric oxide synthase (eNOS) gene has been associated with IR in both non-diabetic and diabetic subjects. Aim of the study was to assess the reciprocal relationships between T−786→C eNOS polymorphism and IR in ischemic and non-ischemic cardiomyopathy.
Method
A group of 132 patients (108 males, median age 65 years) with global left ventricular (LV) dysfunction secondary to ischemic or non-ischemic heart disease was enrolled. Genotyping of T−786→C eNOS gene promoter, fasting glucose, insulin, and insulin resistance (defined as HOMA-IR index > 2.5) were determined in all patients.
Results
Genotyping analysis yielded 37 patients homozygous for the T allele (TT), 70 heterozygotes (TC) and 25 homozygous for C (CC). Patients with CC genotype had significantly higher systemic arterial pressure, blood glucose, plasma insulin and HOMA index levels than TT. At multivariate logistic analysis, the history of hypertension and the genotype were the only predictors of IR. In particular, CC genotype increased the risk of IR (CI% 1.4-15.0, p < 0.01) 4.5-fold. The only parameter independently associated with the extent of LV dysfunction and the presence of heart failure (HF) was the HOMA index (2.4 CI% 1.1-5.6, p < 0.04).
Conclusions
T−786→C eNOS polymorphism was the major independent determinant of IR in a population of patients with ischemic and non-ischemic cardiomyopathy. The results suggest that a condition of primitive eNOS lower expression can predispose to an impairment of glucose homeostasis, which in turn is able to affect the severity of heart disease.
doi:10.1186/1471-2350-13-92
PMCID: PMC3495192  PMID: 23031426
eNOS polymorphism; Insulin resistance; Heart failure
5.  Cobalt-Protoporphyrin Improves Heart Function by Blunting Oxidative Stress and Restoring NO Synthase Equilibrium in an Animal Model of Experimental Diabetes 
Myocardial dysfunction and coronary macro/microvascular alterations are the hallmarks of diabetic cardiomyopathy and are ascribed to increased oxidative stress and altered nitric oxide synthase (NOS) activity. We hypothesize that pre-treatment by cobalt-protoporphyrin IX (CoPP) ameliorates both myocardial function and coronary circulation in streptozotocin (STZ)-induced diabetic rats. Isolated hearts from diabetic rats in Langendorff configuration displayed lower left ventricular function and higher coronary resistance (CR) compared to hearts from control animals. CoPP treatment of diabetic animals (0.3 mg/100 g body weight i.p., once a week for 3 weeks) significantly increased all the contractile/relaxation indexes (p < 0.01), while decreasing CR (p < 0.01). CoPP enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, as indicated by the significant (p < 0.05) decrease in heart % GSSG, O2− and malondialdehyde (MDA) levels. CoPP increased adiponectin levels and phosphorylation of AKT and AMPK and reversed the eNOS/iNOS expression imbalance observed in the untreated diabetic heart. Furthermore, after CoPP treatment, a rise in malonyl-CoA as well as a decrease in acetyl-CoA was observed in diabetic hearts. In this experimental model of diabetic cardiomyopathy, CoPP treatment improved both cardiac function and coronary flow by blunting oxidative stress, restoring eNOS/iNOS expression balance and increasing HO-1 levels, thereby favoring improvement in both endothelial function and insulin sensitivity.
doi:10.3389/fphys.2012.00160
PMCID: PMC3366474  PMID: 22675305
diabetes; cardiac contractility; AMPK; nitric oxide; coronary microcirculation
6.  The Chemical Biology of Nitric Oxide. Implications in Cellular Signaling 
Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the last few years, the importance of steady state NO concentrations have emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose 5 basic distinct concentration levels of NO activity; cGMP mediated processes ([NO] <1–30 nM; Akt phosphorylation ([NO] = 30–100 nM); stabilization of HIF-1α ([NO] = 100–300 nM); phosphorylation of p53 ([NO] > 400 nM) and nitrosative stress (1 µM). In general, lower NO concentrations promote cell survival and proliferation, while higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species (ROS) generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell cycle arrest profile to a cell survival one. The resulting reactive nitrogen species (RNS) that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability which are referred to as Kinetic Determinants for Molecular Target Interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.
doi:10.1016/j.freeradbiomed.2008.03.020
PMCID: PMC2572721  PMID: 18439435
nitric oxide oxidative nitrosative stress
7.  O-linked GlcNAc Modification of Cardiac Myofilament Proteins: A Novel Regulator of Myocardial Contractile Function 
Circulation research  2008;103(12):1354-1358.
In addition to O-Phosphorylation, O-linked modifications of serine and threonine by β-N-acetyl-D-glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O-GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O-GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-Biotin labeling followed by DTT replacement and LC-MS/MS site mapping was adopted. Thirty-two O-GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force-[Ca2+] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force (Fmax) and Hill coefficient (n) were not modified. Using a pan-specific O-GlcNAc antibody it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O-GlcNAcylation. This study provides the first identification of O-GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function.
doi:10.1161/CIRCRESAHA.108.184978
PMCID: PMC2615199  PMID: 18988896
O-GlcNAc; Myofilaments; Post-translational Modifications; Cardiac Contractility; Diabetic Cardiomyopathy

Results 1-7 (7)