LncRNAs and CircRNAs in Endoplasmic Reticulum Stress: A Promising Target for Cardiovascular Disease?

The endoplasmic reticulum (ER) is a principal subcellular organelle responsible for protein quality control in the secretory pathway, preventing protein misfolding and aggregation. Failure of protein quality control in the ER triggers several molecular mechanisms such as ER-associated degradation (ERAD), the unfolded protein response (UPR) or reticulophagy, which are activated upon ER stress (ERS) to re-establish protein homeostasis by transcriptionally and translationally regulated complex signalling pathways. However, maintenance over time of ERS leads to apoptosis if such stress cannot be alleviated. The presence of abnormal protein aggregates results in loss of cardiomyocyte protein homeostasis, which in turn results in several cardiovascular diseases such as dilated cardiomyopathy (DCM) or myocardial infarction (MI). The influence of a non-coding genome in the maintenance of proper cardiomyocyte homeostasis has been widely proven. To date, the impact of microRNAs in molecular mechanisms orchestrating ER stress response has been widely described. However, the role of long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) is just beginning to be addressed given the potential role of these RNA classes as therapeutic molecules. Here, we provide a current state-of-the-art review of the roles of distinct lncRNAs and circRNAs in the modulation of ERS and UPR and their impact in cardiovascular diseases.

The burden of cystic fibrosis in North Africa.

Background: Over 200 pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are associated with cystic fibrosis (CF)-the most prevalent autosomal recessive disease globally, the p.Phe508del variant being the most commonly observed. Main text: Recent epidemiological studies suggest a higher global prevalence of CF than previously thought. Nevertheless, comprehensive CF data remains extremely scarce among African populations, contributing to a significant information gap within the African healthcare system. Consequently, the underestimation of CF among children from African populations is likely. The goal of this article is to review the pathogenesis of CF and its prevalence in the countries of North Africa. Conclusion: The prevalence of CF in North African countries is likely underestimated due to the complexity of the disease and the lack of a timely, proper clinical and genetic investigation that allows the early identification of CF patients and thus facilitates therapeutic recommendations. Therefore, specific genetic and epidemiological studies on African individuals showing CF symptoms should be conducted to enhance the diagnostic yield of CF in Africa.

Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias.

Nav1.5 is the predominant cardiac sodium channel subtype, encoded by the SCN5A gene, which is involved in the initiation and conduction of action potentials throughout the heart. Along its biosynthesis process, Nav1.5 undergoes strict genomic and non-genomic regulatory and quality control steps that allow only newly synthesized channels to reach their final membrane destination and carry out their electrophysiological role. These regulatory pathways are ensured by distinct interacting proteins that accompany the nascent Nav1.5 protein along with different subcellular organelles. Defects on a large number of these pathways have a tremendous impact on Nav1.5 functionality and are thus intimately linked to cardiac arrhythmias. In the present review, we provide current state-of-the-art information on the molecular events that regulate SCN5A/Nav1.5 and the cardiac channelopathies associated with defects in these pathways.

Genetics and Epigenetics of Atrial Fibrillation.

Atrial fibrillation (AF) is known to be the most common supraventricular arrhythmia affecting up to 1% of the general population. Its prevalence exponentially increases with age and could reach up to 8% in the elderly population. The management of AF is a complex issue that is addressed by extensive ongoing basic and clinical research. AF centers around different types of disturbances, including ion channel dysfunction, Ca2+-handling abnormalities, and structural remodeling. Genome-wide association studies (GWAS) have uncovered over 100 genetic loci associated with AF. Most of these loci point to ion channels, distinct cardiac-enriched transcription factors, as well as to other regulatory genes. Recently, the discovery of post-transcriptional regulatory mechanisms, involving non-coding RNAs (especially microRNAs), DNA methylation, and histone modification, has allowed to decipher how a normal heart develops and which modifications are involved in reshaping the processes leading to arrhythmias. This review aims to provide a current state of the field regarding the identification and functional characterization of AF-related epigenetic regulatory networks.

Role of SCN5A coding and non-coding sequences in Brugada syndrome onset: What's behind the scenes?

BACKGROUND: Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia associated with a high risk of sudden cardiac death (SCD) due to ventricular fibrillation (VF). BrS is characterized by coved-type ST-segment elevation in the right precordial leads (V1-V3). Mutations in SCN5A gene coding for the α-subunit of the NaV1.5 cardiac sodium channel are identified in 15-30% of BrS cases. Genetic testing of BrS patients generally involves sequencing of the protein-coding portions and flanking intronic regions of SCN5A. This excludes the 5'UTR and 3'UTR from the routine genetic testing. METHODS: We here screened the coding sequence, the flanking intronic regions as well as the 5' and 3'UTR regions of SCN5A gene and further five candidate genes (GPD1L, SCN1B, KCNE3, SCN4B, and MOG1) in a Tunisian family diagnosed with BrS. RESULTS: A new SCN5A-Q1000K mutation was identified along with two common polymorphisms (H558R and D1819). Multiple genetic variants were identified on the SCN5A 3'UTR, one of which is predicted to create additional microRNA binding site for miR-1270. Additionally, we identified the hsa-miR-219a-rs107822. No relevant coding sequence variant was identified in the remaining studied candidate genes. CONCLUSIONS: The absence of genotype-phenotype concordance within all the identified genetic variants in this family gives extra evidences about the complexity of the disease and suggests that the occurrence and prognosis of BrS is most likely controlled by a combination of multiple genetic factors, rather than a single variant. Most SCN5A variants were localized in non-coding regions hypothesizing an impact on the miRNA-target complementarities.

Pitx2 impairs calcium handling in a dose-dependent manner by modulating Wnt signalling.

AIMS: Atrial fibrillation (AF) is the most common type of arrhythmia in humans, yet the genetic cause of AF remains elusive. Genome-wide association studies (GWASs) have reported risk variants in four distinct genetic loci, and more recently, a meta-GWAS has further implicated six new loci in AF. However, the functional role of these AF GWAS-related genes in AF and their inter-relationship remain elusive. METHODS AND RESULTS: To get further insights into the molecular mechanisms driven by Pitx2, calcium handling and novel AF GWAS-associated gene expression were analysed in two distinct Pitx2 loss-of-function models with distinct basal electrophysiological defects; a novel Pitx2 conditional mouse line, Sox2CrePitx2, and our previously reported atrial-specific NppaCrePitx2 line. Molecular analyses of the left atrial appendage in NppaCrePitx2(+/-) and NppaCrePitx2(-/-) adult mice demonstrate that AF GWAS-associated genes such as Zfhx3, Kcnn3, and Wnt8a are severely impaired but not Cav1, Synpo2l, nor Prrx1. In addition, multiple calcium-handling genes such as Atp2a2, Casq2, and Plb are severely altered in atrial-specific NppaCrePitx2 mice in a dose-dependent manner. Functional assessment of calcium homeostasis further underscores these findings. In addition, multiple AF-related microRNAs are also impaired. In vitro over-expression of Wnt8, but not Zfhx3, impairs calcium handling and modulates microRNA expression signature identified in Pitx2 loss-of-function models. CONCLUSION: Our data demonstrate a dose-dependent relation between Pitx2 expression and the expression of AF susceptibility genes, calcium handling, and microRNAs and identify a complex regulatory network orchestrated by Pitx2 with large impact on atrial arrhythmogenesis susceptibility.

Absence of family history and phenotype-genotype correlation in pediatric Brugada syndrome: more burden to bear in clinical and genetic diagnosis.

Brugada syndrome (BrS) is an autosomal-dominant genetic cardiac disorder caused in 18-30 % of the cases by SCN5A gene mutations and manifested by an atypical right bundle block pattern with ST segment elevation and T wave inversion in the right precordial leads. The syndrome is usually detected after puberty. The identification of BrS in pediatric patients is thus a rare occurrence, and most of the reported cases are unmasked after febrile episodes. Usually, having a family history of sudden death represents the first reason to perform an ECG in febrile children. However, this practice makes the sporadic cases of cardiac disease and specially the asymptomatic ones excluded from this diagnosis. Here, we report a sporadic case of a 2-month-old male patient presented with vaccination-related fever and ventricular tachycardia associated with short breathing, palpitation and cold sweating. ECG changes were consistent with type 1 BrS. SCN5A gene analysis of the proband and his family revealed a set of mutations and polymorphisms differentially distributed among family members, however, without any clear genotype-phenotype correlation. Based on our findings, we think that genetic testing should be pursued as a routine practice in symptomatic and asymptomatic pediatric cases of BrS, with or without family history of sudden cardiac death. Similarly, our study suggests that pediatrician should be encouraged to perform an ECG profiling in suspicious febrile children and quickly manage fever since it is the most important factor unmasking BrS in children.

Regulation of SCN5A by microRNAs: miR-219 modulates SCN5A transcript expression and the effects of flecainide intoxication in mice.

BACKGROUND: The human cardiac action potential in atrial and ventricular cells is initiated by a fast-activating, fast-inactivating sodium current generated by the SCN5A/Nav1.5 channel in association with its ß1/SCN1B subunit. The role of Nav1.5 in the etiology of many cardiac diseases strongly suggests that proper regulation of cell biology and function of the channel is critical for normal cardiac function. Hence, numerous recent studies have focused on the regulatory mechanisms of Nav1.5 biosynthetic and degradation processes as well as its subcellular localization. OBJECTIVE: The purpose of this study was to investigate the role of microRNAs in the Scn5a/Nav1.5 posttranscriptional regulation. METHODS: Quantitative polymerase chain reaction, immunohistochemical and electrophysiological measurements of distinct microRNA gain-of-function experiments in cardiomyocytes for the assessment of Scn5a expression. RESULTS: Functional studies of HL-1 cardiomyocytes and luciferase assays in fibroblasts demonstrate that Scn5a is directly (miR-98, miR-106, miR-200, and miR-219) and indirectly (miR-125 and miR-153) regulated by multiple microRNAs displaying distinct time-dependent profiles. Cotransfection experiments demonstrated that miR-219 and miR-200 have independent opposite effects on Scn5a expression modulation. Of all the microRNAs studied, only miR-219 increases Scn5a expression levels, leading to altered contraction rhythm of HL-1 cardiomyocytes. Electrophysiological analyses in HL-1 cells revealed that miR-219 increases the sodium current. In vivo administration of miR-219 does not alter normal cardiac rhythm, but abolishes some of the effects of flecainide intoxication in mice, particularly QRS prolongation. CONCLUSION: This study demonstrates the involvement of multiple microRNAs in the regulation of Scn5a. Particularly, miR-219 increases Scn5a/Nav1.5 transcript and protein expression. Our data suggest that microRNAs, such as miR-219, constitute a promising therapeutical tool to treat sodium cardiac arrhythmias.

Combined analysis of EPHX1, GSTP1, GSTM1 and GSTT1 gene polymorphisms in relation to chronic obstructive pulmonary disease risk and lung function impairment.

Smoking is considered as the major causal factor of chronic obstructive pulmonary disease (COPD). Nevertheless, a minority of chronic heavy cigarette smokers develops COPD. This suggests important contribution of other factors such as genetic predisposing. Our objective was to investigate combined role of EPHX1, GSTP1, M1 and T1 gene polymorphisms in COPD risk, its phenotypes and lung function impairment. Prevalence of EPHX1, GSTP1, M1 and T1 gene polymorphisms were assessed in 234 COPD patients and 182 healthy controls from Tunisia. Genotypes of EPHX1 (Tyr113His; His139Arg) and GSTP1 (Ile105Val; Ala114Val) polymorphisms were performed by PCR-RFLP, while the deletion in GSTM1 and GSTT1 genes was determined using multiplex PCR. Analysis of combinations showed a significant association of 113His/His EPHX1/null-GSTM1 (OR=4.07) and null-GSTM1/105Val/Val GSTP1 (OR =3.56) genotypes with increased risk of COPD (respectively P=0.0094 and P=0.0153). The null-GSTM1/ null-GSTT1, 105Val/Val GSTP1/null GSTT1, 113His/His EPHX1/null-GSTM1 and null-GSTM1/105Val/Val GSTP1 genotypes were related to emphysema (respectively P=0.01; P=0.009; P=0.008 and P=0.001). Combination of 113His/His EPHX1/null-GSTM1 genotypes showed a significant association with the decrease of Δ FEV1 in patients (P =0.028).In conclusion, our results suggest combined EPHX1, GSTP1, GSTM1 and GSTT1 genetic polymorphisms may play a significant role in the development of COPD, emphysema and decline of the lung function.

PITX2 insufficiency leads to atrial electrical and structural remodeling linked to arrhythmogenesis.

BACKGROUND: Pitx2 is a homeobox transcription factor that plays a pivotal role in early left/right determination during embryonic development. Pitx2 loss-of-function mouse mutants display early embryonic lethality with severe cardiac malformations, demonstrating the importance of Pitx2 during cardiogenesis. Recently, independent genome-wide association studies have provided new evidence for a putative role of PITX2 in the adult heart. These studies have independently reported several risk variants close to the PITX2 locus on chromosome 4q25 that are strongly associated with atrial fibrillation in humans. METHODS AND RESULTS: We show for the first time that PITX2C expression is significantly decreased in human patients with sustained atrial fibrillation, thus providing a molecular link between PITX2 loss of function and atrial fibrillation. In addition, morphological, molecular, and electrophysiological characterization of chamber-specific Pitx2 conditional mouse mutants reveals that atrial but not ventricular chamber-specific deletion of Pitx2 results in differences in the action potential amplitude and resting membrane potential in the adult heart as well as ECG characteristics of atrioventricular block. Lack of Pitx2 in atrial myocardium impairs sodium channel and potassium channel expression, mediated in part by miRNA misexpression. CONCLUSIONS: This study thus identifies Pitx2 as an upstream transcriptional regulator of atrial electric function, the insufficiency of which results in cellular and molecular changes leading to atrial electric and structural remodeling linked to arrhythmogenesis.

Modulation of conductive elements by Pitx2 and their impact on atrial arrhythmogenesis.

The development of the heart is a complex process during which different cell types progressively contribute to shape a four-chambered pumping organ. Over the last decades, our understanding of the specification and transcriptional regulation of cardiac development has been greatly augmented as has our understanding of the functional bases of cardiac electrophysiology during embryogenesis. The nascent heart gradually acquires distinct cellular and functional characteristics, such as the formation of contractile structures, the development of conductive capabilities, and soon thereafter the co-ordinated conduction of the electrical impulse, in order to fulfil its functional properties. Over the last decade, we have learnt about the consequences of impairing cardiac morphogenesis, which in many cases leads to congenital heart defects; however, we are not yet aware of the consequences of impairing electrical function during cardiogenesis. The most prevalent cardiac arrhythmia is atrial fibrillation (AF), although its genetic aetiology remains rather elusive. Recent genome-wide association studies have identified several genetic variants highly associated with AF. Among them are genetic variants located on chromosome 4q25 adjacent to PITX2, a transcription factor known to play a critical role in left-right asymmetry and cardiogenesis. Here, we review new insights into the cellular and molecular links between PITX2 and AF.

MicroRNA profiling during mouse ventricular maturation: a role for miR-27 modulating Mef2c expression.

AIMS: non-coding RNA has been recently demonstrated to be a novel mechanism for modulation of gene expression at the post-transcriptional level. The importance of microRNAs in the cardiovascular system is now apparent. Mutations of distinct microRNAs have provided evidence for fundamental roles of microRNAs during cardiovascular development. However, there is limited information about global microRNA profiles during mouse heart development. In this study, we have gained insight from the expression profiles of microRNAs during mouse ventricular development by microarray and qRT-PCR analysis. METHODS AND RESULTS: our microarray analysis reveals that relatively few microRNAs display either increasing or decreasing expression profiles during ventricular chamber formation. Interestingly, most of the differentially expressed microRNAs display a rather discrete peak of expression at particular developmental stages. Furthermore, we demonstrate that microRNA-27b (miR-27b) displays an overt myocardial expression during heart development and that the transcription factor-encoding gene Mef2c is an miR-27b target. CONCLUSION: our data present a comprehensive profile of microRNA expression during ventricular maturation, providing an entry point for investigation of the functional roles of the most abundantly and differentially expressed microRNAs during cardiogenesis.

Microsomal epoxide hydrolase gene polymorphisms and susceptibility to chronic obstructive pulmonary disease in the Tunisian population.

It is well known that cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD). However, only 10%-20% of chronic heavy cigarette smokers develop symptomatic disease, which suggests the presence of genetic susceptibility. Microsomal epoxide hydrolase (EPHX1) is an enzyme involved in the protective mechanism against oxidative stress. It has been reported that gene polymorphisms of this enzyme may be associated with variations in EPHX1 activity. In this study, we aimed at investigating the relationship between EPHX1 polymorphisms and susceptibility to COPD in the Tunisian population. EPHX1 exon 3 (rs1051740, Tyr113His) and exon 4 (rs2234922, His139Arg) polymorphisms were genotyped by polymerase chain reaction followed by restriction fragment length polymorphism analysis. These techniques were used to examine a total of 416 Tunisian individuals, including 182 blood donors and a group of 234 COPD patients. All subjects were not related. An increased risk for COPD was observed in subjects with EPHX1 His113-His113 genotype (odds ratio = 2.168; confidence interval 1.098-4.283; p = 0.02386). However, multivariate logistic regression analysis showed no significant relationship between the mutant genotype and the disease after adjustment for sex, age, body mass index, smoking status, and pack-year smoking (odds ratio = 1.524; confidence interval, 0.991-6.058; p = 0.06137). Regarding the two subtypes of COPD, our investigations demonstrated that there is no significant correlation between exon 3 polymorphism and the chronic bronchitis subgroup (p = 0.09034). The relation between exon 3 polymorphism and emphysema was significant in the univariate analysis (p = 0.02257), but no association was found after controlling for classic risk factors (p = 0.06273). In conclusion, our results showed that there is a weak relation between 113His genotype and COPD, and no apparent relation between 139Arg and COPD in the studied Tunisian population.

Association of GSTM1 and GSTT1 polymorphisms with chronic obstructive pulmonary disease in a Tunisian population.

GSTM1 and GSTT1 polymorphisms have been proposed in relationship with chronic obstructive pulmonary disease (COPD). We investigated the association between these polymorphisms and COPD (as well as its subtypes emphysema and chronic bronchitis) in 234 COPD patients and 182 healthy controls in the Tunisian population. Genotyping was performed using multiplex PCR. GSTM1-null genotype frequency was significantly higher in COPD patients than in controls (P = 0.02); however, multivariate analysis of cofounding variables showed no independent association with this genotype (P = 0.073). In contrast, the association of the GSTM1-null genotype with emphysema was significant, even after adjustment for risk factors (P = 0.011). There were no significant differences in GSTT1 genotypes between patients and controls. The GSTM1 null allele is likely not an independent risk factor for COPD but is related to emphysema, whereas the GSTT1 gene is not associated with the disease.

Hemoglobinopathies in North Africa: a review.

Hemolytic anemias are very common diseases. Among these diseases, hemoglobinopathies are widely spread throughout the Mediterranean Basin, including North Africa (Tunisia, Algeria and Morocco). Their severity and disabling nature make them a major public health problem. This study includes our data on the Tunisian hemoglobinopathies together with all the reports concerning epidemiological, clinical and molecular aspects in Algerian and Moroccan populations. Investigation methods begin with the application of several techniques for hemoglobin (Hb) analyses [electrophoresis and isoelectric focusing (IEF), micro-chromatography assay] of anemic patients in various hospital departments. Molecular investigation by DNA analyses completes the hematological and biochemical studies using polymerase chain reaction (PCR) followed by enzymatic digestion and/or denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP) and sequencing. These methods offer screening for a large number of families affected by sickle cell disease and thalassemia. In Tunisia, Algeria, and Morocco, more than 45 mutations have been identified on the beta-globin gene. The most common in Tunisia and in Algeria are codon 39 (C>T) and IVS-I-110 (G>A), which together account for more than 50% of all mutations. In Morocco, the predominant mutations are codon 39 and frameshift codon (FSC) 8 (-AA). The identification of molecular defects in the betagene contributes to the development of diagnostic tests (prenatal diagnosis), and gives us the opportunity to help many couples. Our studies of the haplotypes of the beta(S), codon 39 and IVS-I-110 origins allowed the hypothesis of a Benin origin for beta(S), a local North African origin for codon 39 and an Eastern Mediterranean origin for IVS-I-110. The analysis of polymorphisms associated with a moderate phenotype of beta-thalassemia (beta-thal) and sickle cell disease in North Africa has shown, in several cases, a strong association with some mutations and restriction fragment length polymorphisms (RFLP) haplotype IX on the beta-globin locus and the -158 (C>T) polymorphism in 5' on the (G)gamma-globin gene. Finally, more knowledge on the regulation of the beta-globin locus may contribute to the improvement of investigation, monitoring and treatment of hemoglobinopathies.