Hypertension is a major risk factor for cardiovascular diseases and is influenced by both environmental and genetic factors. A large body of evidence supports the contribution of Zinc transporter 7 of the SLC30A7 gene (solute carrier family 30 member 7) located on chromosome 1 and its association with the early onset of essential hypertension. The present study aims (i) to replicate and validate the previously reported associations between the intronic variant rs17123521 of the SLC30A7 gene and hypertension on a sample of precisely defined early-onset hypertensive cases and normotensive controls.

Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in the Unc51-like-kinase 4 (ULK4) gene, to be associated with blood pressure. Aims (i) to replicate and validate the previously reported associations between ULK4 gene and hypertension on hypertensive cases and normotensive controls, (ii) to identify in silico the potential functionality of the associated variants. A total of 558 genotyped Caucasian subjects (n=293 cases & n=265 controls) were recruited and clinically characterized at the Hypertension clinic, University of Ottawa Heart Institute and Institute of Cardiology in Warsaw, Poland. A total of 58 ULK4 SNPs were tested. Our study fails to find any significant association between ULK4 gene and hypertension. However, sex-specific categorization of data revealed strong quantitative association between (I) rs3897976 (p=0.0008) and blood potassium in hypertensive females and (ii) rs9818193 (p=0.0005) and urinary sodium in normotensive female. No signification association were found in males. In-silico motif search revealed differential affinity between the common and the rare allele of rs3897976 and Myocyte enhancer factor-2 (Mef2) and Forkhead box protein O1 (FOXO1) and of rs9818193 and Promyelocytic leukemia zinc finger protein (PLZF) and GATA binding protein 5 (GATA5). Our study unveils novel significant associations between blood potassium levels, urinary sodium levels and ULK4 gene in Caucasian females. Future work needs to confirm the functionality of rs3897976 and rs9818193 polymorphisms.

BACKGROUND:Lamin A/C gene (LMNA) mutations frequently cause cardiac and/or skeletal muscle diseases called striated muscle laminopathies. We created a zebrafish muscular laminopathy model using CRISPR/Cas9 technology to target the zebrafish lmna gene.RESULTS:Heterozygous and homozygous lmna mutants present skeletal muscle damage at 1 day post-fertilization (dpf), and mobility impairment at 4 to 7 dpf. Cardiac structure and function analyses between 1 and 7 dpf show mild and transient defects in the lmna mutants compared to wild type (WT). Quantitative RT-PCR analysis of genes implicated in striated muscle laminopathies show a decrease in jun and nfκb2 expression in 7 dpf homozygous lmna mutants compared to WT. Homozygous lmna mutants have a 1.26-fold protein increase in activated Erk 1/2, kinases associated with striated muscle laminopathies, compared to WT at 7 dpf. Activated Protein Kinase C alpha (Pkc α), a kinase that interacts with lamin A/C and Erk 1/2, is also upregulated in 7 dpf homozygous lmna mutants compared to WT.CONCLUSIONS:This study presents an animal model of skeletal muscle laminopathy where heterozygous and homozygous lmna mutants exhibit prominent skeletal muscle abnormalities during the first week of development. Furthermore, this is the first animal model that potentially implicates Pkc α in muscular laminopathies.

A single nucleotide polymorphism (SNP) in ubiquitin protein ligase NEDD4-Like (NEDD4L) gene (rs4149601 G/A) was identified to have both functional and genetic bases for hypertension. This polymorphism has been well-characterized, but inconsistently replicated. Therefore, the purpose of this study was to replicate the association between the rs4149601 SNP and essential hypertension with a sample of precisely phenotyped early onset hypertensive cases and normotensive controls by performing a casecontrol study.

BACKGROUND High blood pressure is a major risk factor for cardiovascular diseases. Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in the Unc51-like-kinase 4 (ULK4) gene, to be associated with blood pressure. The present study aims to replicate and validate the previously reported associations between ULK4 gene and hypertension on a sample of precisely defined early onset hypertensive cases and normotensive controls. METHODS AND RESULTS The association was evaluated in 558 genotyped Caucasian subjects (n=293cases and n=265 controls) recruited and clinically characterized at the Hypertension clinic at the University of Ottawa Heart Institute, and the Institute of Cardiology in Warsaw, Poland. Blood pressure was measured by 24hr ambulatory blood pressure monitoring. A total of 58 SNPs were tested in association with hypertension. Strategic categorization of data revealed a strong quantitative association between (i) rs3897976 (p= 0.0008,) and blood potassium in female (cases) and pooled data (case and controls), (ii) rs4016425(p= 0.0001) and Urinary creatinine in Caucasian females. Whereas, a protective effect against urinary sodium and rs9818193 (0.0005) was seen in female (controls), Urinary Potassium and rs11129908 (p= 0.0001), rs1691974 (p= 0.0003), rs6780145(p= 0.0003), rs1795348(p= 0.0003), rs2029254 (p=0.0004), rs9818193 (p= 0.0008), rs9876441(p=0.0008), rs12638414 (p=0.0008), rs6599155(p= 0.0009), rs9841301(p=0.0012) in normotensives. All the associations remained significant on the application of stringent multiple corrections. CONCLUSION Our study unveils significant associations between blood potassium, urinary creatinine, urinary sodium, urine potassium and ULK4 gene. To the best of our knowledge these associations have not been reported previously. The significant associations found for urinary creatinine and blood potassium in hypertensive females in this study, may represent a genetic predisposition towards the risk of developing kidney or muscle diseases later in their life. On the other hand, a strong protective effect against urinary sodium and urinary potassium seems to protect and lower the risk of developing cardiovascular and kidney diseases in normotensives and normotensive females. The future work needs to identify the functionality of these SNPs to confirm their role in blood pressure regulation. High blood pressure is a major risk factor for cardiovascular diseases. Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in the Unc51-like-kinase 4 (ULK4) gene, to be associated with blood pressure. The present study aims to replicate and validate the previously reported associations between ULK4 gene and hypertension on a sample of precisely defined early onset hypertensive cases and normotensive controls. The association was evaluated in 558 genotyped Caucasian subjects (n=293cases and n=265 controls) recruited and clinically characterized at the Hypertension clinic at the University of Ottawa Heart Institute, and the Institute of Cardiology in Warsaw, Poland. Blood pressure was measured by 24hr ambulatory blood pressure monitoring. A total of 58 SNPs were tested in association with hypertension. Strategic categorization of data revealed a strong quantitative association between (i) rs3897976 (p= 0.0008,) and blood potassium in female (cases) and pooled data (case and controls), (ii) rs4016425(p= 0.0001) and Urinary creatinine in Caucasian females. Whereas, a protective effect against urinary sodium and rs9818193 (0.0005) was seen in female (controls), Urinary Potassium and rs11129908 (p= 0.0001), rs1691974 (p= 0.0003), rs6780145(p= 0.0003), rs1795348(p= 0.0003), rs2029254 (p=0.0004), rs9818193 (p= 0.0008), rs9876441(p=0.0008), rs12638414 (p=0.0008), rs6599155(p= 0.0009), rs9841301(p=0.0012) in normotensives. All the associations remained significant on the application of stringent multiple corrections. Our study unveils significant associations between blood potassium, urinary creatinine, urinary sodium, urine potassium and ULK4 gene. To the best of our knowledge these associations have not been reported previously. The significant associations found for urinary creatinine and blood potassium in hypertensive females in this study, may represent a genetic predisposition towards the risk of developing kidney or muscle diseases later in their life. On the other hand, a strong protective effect against urinary sodium and urinary potassium seems to protect and lower the risk of developing cardiovascular and kidney diseases in normotensives and normotensive females. The future work needs to identify the functionality of these SNPs to confirm their role in blood pressure regulation.

Mutations in the lamin A/C gene are variably phenotypically expressed; however, it is unclear whether circulating cardiac biomarkers are helpful in the detection and risk assessment of cardiolaminopathies. We sought to assess (1) clinical characteristics including serum biomarkers: high sensitivity troponin T (hsTnT) and N-terminal prohormone brain natriuretic peptide (NT-proBNP) in clinically stable cardiolaminopathy patients, and (2) outcome among pathogenic/likely pathogenic lamin A/C gene (LMNA) mutation carriers. Our single-centre cohort included 53 patients from 21 families. Clinical, laboratory, follow-up data were analysed. Median follow-up was 1522 days. The earliest abnormality, emerging in the second and third decades of life, was elevated hsTnT (in 12% and in 27% of patients, respectively), followed by the presence of atrioventricular block, heart failure, and malignant ventricular arrhythmia (MVA). In patients with missense vs. other mutations, we found no difference in MVA occurrence and, surprisingly, worse transplant-free survival. Increased levels of both hsTnT and NT-proBNP were strongly associated with MVA occurrence (HR > 13, p ≤ 0.02 in both) in univariable analysis. In multivariable analysis, NT-proBNP level > 150 pg/mL was the only independent indicator of MVA. We conclude that assessment of circulating cardiac biomarkers may help in the detection and risk assessment of cardiolaminopathies.

Striated muscle laminopathies are cardiac and skeletal muscle conditions caused by mutations in the lamin A/C gene (LMNA). LMNA codes for the A-type lamins, which are nuclear intermediate filaments that maintain the nuclear structure and nuclear processes such as gene expression. Protein kinase C alpha (PKC-α) interacts with lamin A/C and with several lamin A/C partners involved in striated muscle laminopathies. To determine PKC-α's involvement in muscular laminopathies, PKC-α's localization, activation, and interactions with the A-type lamins were examined in various cell types expressing pathogenic lamin A/C mutations. The results showed aberrant nuclear PKC-α cellular distribution in mutant cells compared to WT. PKC-α activation (phos-PKC-α) was decreased or unchanged in the studied cells expressing LMNA mutations, and the activation of its downstream targets, ERK 1/2, paralleled PKC-α activation alteration. Furthermore, the phos-PKC-α-lamin A/C proximity was altered. Overall, the data showed that PKC-α localization, activation, and proximity with lamin A/C were affected by certain pathogenic LMNA mutations, suggesting PKC-α involvement in striated muscle laminopathies.

Background: To better understand the potential alteration of muscle bioenergetic metabolism by the obesogenic toxicant mono-(2ethylhexyl) phthalate (MEHP) the objectives of this research were to determine the: 1) association between urinary MEHP levels and plasma fatty acid levels in women with obesity who participated in National Health and Nutrition Examination Survey (NHANES) studies, and 2) in vitro effects of MEHP on fatty acid, or glucose supported mitochondrial energetics in C2C12 muscle cells.Results: The association between urinary MEHP from NHANES participants with plasma fatty acid levels was studied via secondary data statistical analyses. 14C-palmitic acid oxidation, Seahorse fatty acid oxidation and glycolysis stress tests and western blot analyses were conducted on C2C12 cells exposed to increasing MEHP concentrations. Increased urinary MEHP in women with obesity was associated with increased plasma gamma-linolenic and arachidonic acid levels. C2C12 myotubes exposed to increasing MEHP concentrations, displayed decreased fatty acid oxidation and mitochondrial bioenergetics. Acyl-CoA synthetase long chain 5 (ACSL5) protein level was also upregulated with increasing MEHP exposure in C2C12 myoblasts. Glycolysis was not significantly modified with increased exposure of C2C12 cells to MEHP.Conclusions: MEHP exposure may alter fatty acid utilization at the whole-body level in women with obesity and fatty acid utilization in muscle cells. Our findings are consistent with the idea that women with obesity may be particularly susceptible to the effects of MEHP, which alters fatty acid metabolism in muscle cells.

The lamin A/C (LMNA) gene codes for nuclear intermediate filaments constitutive of the nuclear lamina. LMNA has 12 exons and alternative splicing of exon 10 results in two major isoforms—lamins A and C. Mutations found throughout the LMNA gene cause a group of diseases collectively known as laminopathies, of which the type, diversity, penetrance and severity of phenotypes can vary from one individual to the other, even between individuals carrying the same mutation. The majority of the laminopathies affect cardiac and/or skeletal muscles. The underlying molecular mechanisms contributing to such tissue-specific phenotypes caused by mutations in a ubiquitously expressed gene are not yet well elucidated. This review will explore the different phenotypes observed in established models of striated muscle laminopathies and their respective contributions to advancing our understanding of cardiac and skeletal muscle-related laminopathies. Potential future directions for developing effective treatments for patients with lamin A/C mutation-associated cardiac and/or skeletal muscle conditions will be discussed.

BACKGROUND:Acyl-CoA Synthetase Long Chain 5 (ACSL5) gene's rs2419621 T/C polymorphism was associated with ACSL5 mRNA expression and response to lifestyle interventions. However, the mechanistic understanding of the increased response in T allele carriers is lacking. Study objectives were to investigate the effect of rs2419621 genotype and ACSL5 human protein isoforms on fatty acid oxidation and respiration.METHODS:Human ACSL5 overexpression in C2C12 mouse myoblasts was conducted to measure 14C palmitic acid oxidation and protein isoform localization in vitro. 14C palmitic acid oxidation studies and Western blot analysis of ACSL5 proteins were carried out in rectus abdominis primary myotubes from 5 rs2419621 T allele carriers and 4 non-carriers. In addition, mitochondrial high-resolution respirometry was conducted on vastus lateralis muscle biopsies from 4 rs2419621 T allele carriers and 4 non-carriers. Multiple linear regression analysis was conducted to test the association between rs2419621 genotype and respiratory quotient related pre- and post-lifestyle intervention measurements in postmenopausal women with overweight or obesity.RESULTS:In comparison to rs2419621 non-carriers, T allele carriers displayed higher levels of i) 683aa ACSL5 isoform, localized mainly in the mitochondria, playing a greater role in fatty acid oxidation in comparison to the 739aa protein isoform ii) in vitro CO2 production in rectus abdominis primary myotubes iii) in vivo fatty acid oxidation and lower carbohydrate oxidation post-intervention iv) ex vivo complex I and II tissue respiration in vastus lateralis muscle.CONCLUSIONS:These results support the conclusion that rs2419621 T allele carriers, are more responsive to lifestyle interventions partly due to an increase in the short ACSL5 protein isoform, increasing cellular, tissue and whole-body fatty acid utilization. With the increasing effort to develop personalized medicine to combat obesity, our findings provide additional insight into genotypes that can significantly affect whole body metabolism and response to lifestyle interventions.

Genetic studies on Acyl-CoA Synthetase Long-Chain 5 (ACSL5) demonstrate an association between rs2419621 genotype and rate of weight loss in women with obesity in response to caloric restriction. Our objectives were to (1) confirm results in two different populations of women with overweight and obesity (2) study rs2419621’s influence on body composition parameters of women with overweight and obesity following lifestyle interventions.

Cellular fatty acid transport is reported to influence body weight loss following a weight loss intervention. The Acyl-CoA Synthetase Long Chain 5 (ACSL5) gene, coding for a transmembrane protein involved in the conversion of fatty acids into fatty acyl-coA esters, produces long and short ACSL5 isoforms by alternative splicing. Previously, the rs2419621 polymorphism has been shown to influence ACSL5 level of expression in skeletal muscle and to be associated with weight loss. This functional polymorphism creates a new E-box recognized by transcription factor MyoD, increasing transcription of the downstream reporter gene. We hypothesize that Caucasian overweight/obese women carrying the rs2419621 rare [T] allele will display healthier body composition parameters, in response to weight loss interventions as compared to wildtype allele carriers. A secondary analysis was performed using the data of obese (BMI >30) and overweight (BMI >25) women ages 46–70 from the Montréal New Emerging Team (MONET n=137) study and Complications Associated with Obesity (CAO n=37) study. Using a multiple linear regression model, statistical associations between rs2419621 genotype and changes in body composition indices were studied. We found that the ACSL5 rs2419621 rare allele was associated with a decrease in total % fat mass (–2.1585, SE=1.098, p=0.0517) and visceral fat (–6.216, SE=2.886, p=0.033) and an increase in % lean mass (2.073, SE=0.985, p=0.037) and muscle attenuation (1.170, SE=0.587, p=0.048) in response to the weight loss interventions. These results indicate that individuals carrying the ACSL5 polymorphism rs2419621 [T] allele were more responsive to lifestyle interventions in comparison to non-carriers.

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplant. Mutations in 60 genes have been associated with DCM. Approximately 6% of all DCM cases are caused by mutations in the lamin A/C gene (LMNA). LMNA codes for type-V intermediate filaments that support the structure of the nuclear membrane and are involved in chromatin structure and gene expression. Most LMNA mutations result in striated muscle diseases while the rest affects the adipose tissue, peripheral nervous system, multiple tissues or lead to progeroid syndromes/overlapping syndromes. Patients with LMNA mutations exhibit a variety of cellular and physiological phenotypes. This paper explores the current phenotypes observed in LMNA-caused DCM, the results and implications of the cellular and animal models of DCM and the prevailing theories on the pathogenesis of laminopathies.

Background LMNA mutations are most frequently involved in the pathogenesis of dilated cardiomyopathy with conduction disease. The goal of this study was to identify LMNA mutations, estimate their frequency among Polish dilated cardiomyopathy patients and characterize their effect both in vivo and in vitro . Methods Between January, 2008 and June, 2012 two patient populations were screened for the presence of LMNA mutations by direct sequencing: 66 dilated cardiomyopathy patients including 27 heart transplant recipients and 39 dilated cardiomyopathy patients with heart failure referred for heart transplantation evaluation, and 44 consecutive dilated cardiomyopathy patients, referred for a family evaluation and mutation screening. Results We detected nine non-synonymous mutations including three novel mutations: p.Ser431*, p.Val256Gly and p.Gly400Argfs*11 deletion. There were 25 carriers altogether in nine families. The carriers were mostly characterized by dilated cardiomyopathy and heart failure with conduction system disease and/or complex ventricular arrhythmia, although five were asymptomatic. Among the LMNA mutation carriers, six underwent heart transplantation, fourteen ICD implantation and eight had pacemaker. In addition, we obtained ultrastructural images of cardiomyocytes from the patient carrying p.Thr510Tyrfs*42. Furthermore, because the novel p.Val256Gly mutation was found in a sporadic case, we verified its pathogenicity by expressing the mutation in a cellular model. Conclusions In conclusion, in the two referral centre populations, the screening revealed five mutations among 66 heart transplant recipients or patients referred for heart transplantation (7.6%) and four mutations among 44 consecutive dilated cardiomyopathy patients referred for familial evaluation (9.1%). Dilated cardiomyopathy patients with LMNA mutations have poor prognosis, however considerable clinical variability is present among family members.

Lamin A/C (LMNA) gene mutations cause dilated cardiomyopathy, often accompanied by conduction disturbances. Our aim was to search for LMNA mutations in individuals with atrial fibrillation.A cohort of Polish subjects (N = 103) with non-valvular atrial fibrillation with a high (48.5%) prevalence of conduction system disturbances was screened for LMNA variants by direct DNA sequencing.We found a single non-synonymous variant (Thr528Met) in a 72-year-old patient with normal left ventricular function and episodes of advanced atrioventricular block. One of his two mutation-carrying daughters had episodes of type I second-degree atrioventricular block on a 24-hour Holter ECG and peak exercise arrhythmia. Interpretation of cardiac anomalies observed in the other daughter was complicated by thyroid insufficiency. A Thr528Met weak pathogenic effect was supported by transient transfections of C2C12 mouse myoblasts and computationally. Another interesting variant was Ile26Ile (c.78C>T), found in a New York Heart Association class III patient with a depressed left ventricular ejection fraction (30%), left bundle branch block, and a family history of heart disease. Ile26Ile was absent in 246 healthy individuals and was computationally predicted to interfere with splicing.LMNA mutations are not a frequent cause of atrial fibrillation even when conduction disease is present. Unlike the majority of LMNA mutations clearly associated with a severe clinical phenotype and a poor prognosis, Thr528Met results in a more subtle pathogenic effect, while Ile26Ile should be considered as a variant of unknown significance.

Introduction: Individual blood pressure (BP) response to high salt intake varies from minimal to substantial and is largely genetically determined. More than 50% of hypertensive patients are salt sensitive (SS) and respond to high salt intake by increasing their BP compared to only 10% in the normotensive population. ENaC is involved in sodium transport in the distal nephrons of the kidney as well as in the CNS in the choroid plexus and hypothalamic neurons. In turn, ENaC is controlled by the E3 ubiquitin ligase protein - NEDD4L. The functional NEDD4L SNP rs4149601 (G/A) influences the ability of NEDD4L to regulate ENaC expression and is associated with high blood pressure and salt sensitivity. We hypothesize that NEDD4L rs4149601 genotype is an independent predictor of BP response to salt intake in hypertensives. Methods: Daytime BP was measured using 24hr ABPM in 191 young (<60 years old) Caucasian hypertensives (BP ≥130/85 mmHg). 24hr urine Na and K + , blood Na + and K + , and BMI were collected. Patient genotyping for the rs4149601 (G/A) SNP was done using TaqMan MGB probe based RT PCR. Linear regression analyzed (SAS Software) the influence of urine Na + , urine K + , blood K + ,blood Na + , age, gender, and BMI on blood pressure traits. Results: In hypertensive rs4149601 G carriers (n=169), systolic BP (SBP) varied significantly with salt intake (p=0.0088) while diastolic BP did not (p=0.7198). An SBP increase of 2.34 mmHg per 50 mmol Na was observed when urine Na alone is considered. Age (p=0.0002) and blood K + (p=0.0466) were also significant determinants of SBP in patients with the G allele. Only blood K + was a significant predictor of diastolic BP (DBP) in hypertensive G allele carriers. None of the factors tested influenced either SBP or DBP in hypertensive patients who are carriers of the A allele. Conclusion: The functional NEDD4L rs4149601 SNP influences individual daytime BP response to salt intake. This result supports the hypothesis that NEDD4L rs4149601 G carriers with intact C2 domain express higher levels of ENaC and therefore are more sensitive to salt intake. Thus, NEDD4L rs4149601 polymorphism genotyping may allow for identification of hypertensive individuals who will benefit to a greater extent from reducing the amount of salt in their diet.

The present study was designed to investigate the impact of exercise training on lipogenic gene expression in liver and lipid partitioning following the ingestion of a high fructose load. Female rats were exercise-trained for 8 wk or kept sedentary before being submitted to a fasting/refeeding protocol. Rats were further subdivided as follow: rats were fasted for 24 h, refed a standard diet for 24 h, starved for another 24 h, and refed with a standard or a high-fructose diet 24 h before sacrifice. Fructose refeeding was associated with an increase in hepatic lipid content, endocannabinoid receptor 1, sterol regulatory element-binding protein1c, and stearoyl-CoA desaturase1 gene expression in both Sed and TR rats. However, desaturation indexes measured in liver (C16 : 1/C16 : 0 and C18 : 1/C18 : 0) and plasma (C18 : 1/C18 : 0) were higher (P<0.01) in TR than in Sed rats following fructose refeeding. It is concluded that exercise training does not significantly affect fat accumulation and the molecular expression of genes involved in lipogenesis after fasting and fructose refeeding but does modify the partitioning of lipids so as to provide more unsaturated fatty acids in liver without affecting liver fat content.

A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo) to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C) and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna(H222P/H222P) mouse model). In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies.