Targeted disruption of the Kcnj5 gene in the female mouse lowers aldosterone levels.

Aldosterone is released from adrenal zona glomerulosa (ZG) cells and plays an important role in Na and K homoeostasis. Mutations in the human inwardly rectifying K channel CNJ type (KCNJ) 5 (KCNJ5) gene encoding the G-coupled inwardly rectifying K channel 4 (GIRK4) cause abnormal aldosterone secretion and hypertension. To better understand the role of wild-type (WT) GIRK4 in regulating aldosterone release, we have looked at aldosterone secretion in a Kcnj5 knockout (KO) mouse. We found that female but not male KO mice have reduced aldosterone levels compared with WT female controls, but higher levels of aldosterone after angiotensin II (Ang-II) stimulation. These differences could not be explained by sex differences in aldosterone synthase (Cyp11B2) gene expression in the mouse adrenal. Using RNAseq analysis to compare WT and KO adrenals, we showed that females also have a much larger set of differentially expressed adrenal genes than males (395 compared with 7). Ingenuity Pathway Analysis (IPA) of this gene set suggested that peroxisome proliferator activated receptor (PPAR) nuclear receptors regulated aldosterone production and altered signalling in the female KO mouse, which could explain the reduced aldosterone secretion. We tested this hypothesis in H295R adrenal cells and showed that the selective PPARα agonist fenofibrate can stimulate aldosterone production and induce Cyp11b2. Dosing mice in vivo produced similar results. Together our data show that Kcnj5 is important for baseline aldosterone secretion, but its importance is sex-limited at least in the mouse. It also highlights a novel regulatory pathway for aldosterone secretion through PPARα that may have translational potential in human hyperaldosteronism

Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases

A missense TGFB2 variant p.(Arg320Cys) causes a paradoxical and striking increase in aortic TGFB1/2 expression.

Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder with a range of cardiovascular, skeletal, craniofacial and cutaneous manifestations. LDS type 4 is caused by mutations in TGFβ ligand 2 (TGFB2) and based on the family pedigrees described to date, appears to have a milder clinical phenotype, often presenting with isolated aortic disease. We sought to investigate its molecular basis in a new pedigree. We identified a missense variant p.(Arg320Cys) (NM_003238.3) in a highly evolutionary conserved region of TGFB2 in a new LDS type 4 pedigree with multiple cases of aortic aneurysms and dissections. There was striking upregulation of TGFB1 and TGFB2 expression on immunofluorescent staining, and western blotting of the aortic tissue from the index case confirming the functional importance of the variant. This case highlights the striking paradox of predicted loss-of-function mutations in TGFB2 causing enhanced TGFβ signaling in this emerging familial aortopathy.Raya Al Maskari has a PhD studentship funded by the Omani government.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ejhg.2016.14

Large Artery Stiffness: Genes and Pathways

Aortic stiffness underlies systolic hypertension, promotes heart failure and is associated with in- creased cardiovascular morbidity and mortality. It is regarded as a primary driver of left ventricular hypertrophy and aortic aneurysms and is linked to the pathogenesis of cognitive impairment, stroke and renal failure. Like most cardiovascular traits, aortic stiffness is a complex trait and is moderately heritable, yet the precise molecular mechanisms that underpin the stiffening process remain poorly defined. This study aimed to employ multiple approaches to further identify the genetic basis of aortic stiffness in a large repository of human donor aortas that had undergone ex vivo pulse wave velocity (PWV) phenotyping. The first part of this work sought to investigate the molecular basis of Loeys-Dietz type 4 syn- drome in a pedigree with multiple cases of aortic aneurysms and dissections. A missense variant p.(Arg320Cys) was identified in a highly evolutionary conserved region of TGFB2. There was strik- ing upregulation of TGFB1, TGFB2 and pSMAD2/3 on imunocytochemical straining and western blotting of the aortic tissue from the index case confirming the functional importance of the variant. This case highlighted the striking paradox of predicted loss-of-function mutations in TGFB2 causing enhanced TGFβ signalling in this emerging familial aortopathy and underscored the significance of TGFβ signalling in aortic extracellular matrix biology. The second part of this work attempted to characterise the biological basis for the susceptibility lo- cus identified in the most recent genome wide analysis of carotid-femoral PWV. While the locus lies within the 14q32.2 gene desert, it contains regulatory elements, with the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) and non-coding RNA DB129663 representing potential targets for these enhancers. The association of five lead SNPs from the genome-wide association studies (GWAS) meta-analysis was examined for ex vivo aortic stiffness and BCL11B and DB129663 aortic mRNA expression. Three of the five SNPs associated significantly with PWV and showed allele- specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B suggesting a protective role for BCL11B. Despite the strong association, BCL11B protein was not detected in the human aorta; however, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. In contrast, DB129663 transcripts were detected in 55% of the samples, and of the five SNPs only one showed allele-specific differences in aortic DB129663 transcripts. No significant differences were observed in PWV between samples expressing or lack- ing DB129663, and therefore the implication of this lncRNA in aortic stiffness remains elusive. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association with PWV. For the final part of this work genetic associations with aortic stiffness were explored in a candidate gene-based study utilising tagging SNPs to effectively capture the genetic information from linkage disequilibrium blocks. Association analyses were performed in young, healthy ENIGMA study par- ticipants selected for high and low PWV values then validated in the remaining ENIGMA cohorts. The association of four lead SNPs was then examined for ex vivo aortic stiffness in human donor aortas. The tissue expression of these SNPs and their encoded proteins was also explored. Neither the aggrecan nor the fibulin-1 SNPs showed significant associations with ex vivo PWV in the donor aortas. The exonic aggrecan tagSNP rs2882676 displayed differential transcript abundance between homozygous allele carriers but this did not translate at the protein level. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan, reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening.Government of the Sultanate of Oma

Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness.

The recent genome-wide analysis of carotid-femoral pulse wave velocity (PWV) identified a significant locus within the 14q32.2 gene desert. Gene regulatory elements for the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) are within this locus and an attractive target for the gene association. We investigated the functional impact of these gene desert SNPs on BCL11B transcript in human aorta to characterize further its role in aortic stiffness. To do this, we used a large repository of aortic tissues (n = 185) from an organ transplant program and assessed ex vivo stiffness of the aortic rings. We tested association of three lead SNPs from the GWAS meta-analysis with ex vivo aortic stiffness and BCL11B aortic mRNA expression: rs1381289 and rs10782490 SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B expression, suggesting a protective role for BCL11B. Despite strong association, we could not detect BCL11B protein in the human aorta. However, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. Our data confirm the significance of the 14q32.2 region as a risk locus for aortic stiffness and an upstream regulator of BCL11B. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association of BCL11B with aortic stiffness.This work was funded by a PhD studentship for RAM from the Omani Government, Rosetrees Trust (Ref. CM374), British Heart Foundation (FS12/8/29377) and the UK NIHR

Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness.

The recent genome-wide analysis of carotid-femoral pulse wave velocity (PWV) identified a significant locus within the 14q32.2 gene desert. Gene regulatory elements for the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) are within this locus and an attractive target for the gene association. We investigated the functional impact of these gene desert SNPs on BCL11B transcript in human aorta to characterize further its role in aortic stiffness. To do this, we used a large repository of aortic tissues (n = 185) from an organ transplant program and assessed ex vivo stiffness of the aortic rings. We tested association of three lead SNPs from the GWAS meta-analysis with ex vivo aortic stiffness and BCL11B aortic mRNA expression: rs1381289 and rs10782490 SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B expression, suggesting a protective role for BCL11B. Despite strong association, we could not detect BCL11B protein in the human aorta. However, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. Our data confirm the significance of the 14q32.2 region as a risk locus for aortic stiffness and an upstream regulator of BCL11B. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association of BCL11B with aortic stiffness.This work was funded by a PhD studentship for RAM from the Omani Government, Rosetrees Trust (Ref. CM374), British Heart Foundation (FS12/8/29377) and the UK NIHR

The matrix proteins aggrecan and fibulin-1 play a key role in determining aortic stiffness

Stiffening of the aorta is an important independent risk factor for myocardial infarction and stroke. Yet its genetics is complex and little is known about its molecular drivers. We have identified for the first time, tagSNPs in the genes for extracellular matrix proteins, aggrecan and fibulin-1, that modulate stiffness in young healthy adults. We confirmed SNP associations with ex vivo stiffness measurements and expression studies in human donor aortic tissues. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening