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Scott Wilson,1,2 Andrea Bowyer,3 Stephen B Harrap4 1Department of Renal Medicine, The Alfred Hospital, 2Baker IDI, Melbourne, 3Department of Anaesthesia, Royal Melbourne Hospital, 4University of Melbourne, Parkville, VIC, Australia Abstract: The clinical characterization of cardiovascular dynamics during hemodialysis (HD) has important pathophysiological implications in terms of diagnostic, cardiovascular risk assessment, and treatment efficacy perspectives. Currently the diagnosis of significant intradialytic systolic blood pressure (SBP) changes among HD patients is imprecise and opportunistic, reliant upon the presence of hypotensive symptoms in conjunction with coincident but isolated noninvasive brachial cuff blood pressure (NIBP) readings. Considering hemodynamic variables as a time series makes a continuous recording approach more desirable than intermittent measures; however, in the clinical environment, the data signal is susceptible to corruption due to both impulsive and Gaussian-type noise. Signal preprocessing is an attractive solution to this problem. Prospectively collected continuous noninvasive SBP data over the short-break intradialytic period in ten patients was preprocessed using a novel median hybrid filter (MHF) algorithm and compared with 50 time-coincident pairs of intradialytic NIBP measures from routine HD practice. The median hybrid preprocessing technique for continuously acquired cardiovascular data yielded a dynamic regression without significant noise and artifact, suitable for high-level profiling of time-dependent SBP behavior. Signal accuracy is highly comparable with standard NIBP measurement, with the added clinical benefit of dynamic real-time hemodynamic information. Keywords: continuous monitoring, blood pressure

Objectives: Genes implicated in monogenic human cardiac hypertrophy (CH) might also be involved in the more common polygenic forms. The Hypertrophic Heart Rat (HHR) is a unique normotensive model of spontaneous polygenic ventricular hypertrophy leading to cardiac failure and premature death. Our aim was to survey mRNA expression and genetic variants in genes previously associated with monogenic forms of dilated and hypertrophic cardiomyopathy in humans utilising the HHR.Design and methods: We used Affymetrix GeneChip® Rat Gene 1.0 ST arrays to measure whole-genome mRNA in left ventricles of HHR and its normal control, the Normal Heart Rat (NHR) (n=8 per group). We also performed whole-genome DNA sequencing of both strains using the HiSeq 2000 platform. We aligned and compared sequences to the Rat Genome Database v3.4 and identified four types of variants: SNPs, insertions and deletions (InDels), copy number variations (CNVs) and structural variants (SVs).Results: We found 15 (Cryab, Dsg2, Lama4, Mybpc3, Myh6, Myh7,Myl2, Nexn, Psen2, Rbm20, Tnnc1, Tnni3, Tnnt2, Tpm1, Ttn) of the initial known 40 candidate genes significantly differentially expressed in the HHR (FDR<0.05). Their fold change was small (∼1.3) consistent with a polygenic contribution. Overall, we found 182 variants unique to the HHR in our gene subset. Most variants were SNPs (n=101), followed by InDels (n=67), CNVs (n=12) and SVs (n=2). No major mutations typical of human monogenic cardiomyopathies were found. The majority of SNPs are located in non-coding regions (n=98) except for two variants in the coding region for the gene for troponin T type 2 (Tnnt2) and one in its 3' untranslated region. Interestingly, both mutations in the coding region are synonymous and do not cause a change in the amino acid or protein sequence. Although the variants found in gene for Tnnt2 are synonymous, silent mutations may alter gene expression levels by transfer RNA availability and influence phenotypically variability.Conclusions: Our study is the first to show that genes involved in monogenic human forms of hypertrophy may also contribute through changed expression and subtle variants in DNA sequence to the common polygenic form of left ventricular hypertrophy. [ABSTRACT FROM AUTHOR]

Objective: Cardiac hypertrophy (CH) is the main risk factor for heart disease after age. Multiple genetic factors are known to be involved but still poorly understood. We have previously described a quantitative trait locus (QTL) cardiac mass 22 (Cm22) on chromosome 2 which influences heart size independently of blood pressure. Our aim was to determine which genes in QTL Cm22 contribute to CH using a polygenic genetic model.Design and method: We used a genomic and transcriptomic approach to identify genes in Cm22 contributing to CH. Firstly, we used the Illumina HiSeq 2000 platform to sequence the whole-genome of the Hypertrophic Heart Rat (HHR), a normotensive genetic model of CH, and its control, the Normal Heart Rat (NHR). Both genomes were aligned and compared to Rat Genome Database v3.4 and four types of variants were analysed: single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), copy number variations (CNVs) and structural variants (SVs). Secondly, we investigated gene expression of all Cm22 genes in left ventricles of HHR and NHR (n=8 per group) using Affymetrix GeneChip® Rat Gene 1.0 ST arrays. We then combined these data to identify unique variants in genes differentially expressed in QTL Cm22.Results: Overall, approximately 5.7 and 5 million variants are present in HHR and NHR, respectively, and the majority of the variants (80%) were SNPs. In QTL Cm22, we found approximately 10 thousand variants in each strain. The array data showed that gene for tripartite motif-containing 55 (Trim55) was significantly downregulated in HHR (FC=-2.08; FDR=0.049). We validated these findings by real-time polymerase chain reaction (qPCR), which showed that Trim55 mRNA is down-regulated from neonatal to late adulthood in the CH model.Analysis of the gene region of Trim55 identified 12 and 24 variants in HHR and NHR, respectively. One missense mutation in the HHR was located in its ninth exon. In silico studies demonstrated that this mutation may cause a change in the protein structure which may affect function.Conclusion: The Trim55 gene in QTL CM22 is a novel candidate gene for polygenic hypertrophy development independent of blood pressure. [ABSTRACT FROM PUBLISHER]

Aims: Prevention of human hypertension is an important challenge and has been achieved in experimental models. Brief treatment with renin-angiotensin system (RAS) inhibitors permanently reduces the genetic hypertension of the spontaneously hypertensive rat (SHR). The kidney is involved in this fascinating phenomenon, but relevant changes in gene expression are unknown., Methods and Results: In SHR, we studied the effect of treatment between 10 and 14 weeks of age with the angiotensin receptor blocker, losartan, or the angiotensin-converting enzyme inhibitor, perindopril [with controls for non-specific effects of lowering blood pressure (BP)], on differential RNA expression, DNA methylation, and renin immunolabelling in the kidney at 20 weeks of age. RNA sequencing revealed a six-fold increase in renin gene (Ren) expression during losartan treatment (P < 0.0001). Six weeks after losartan, arterial pressure remained lower (P = 0.006), yet kidney Ren showed reduced expression by 23% after losartan (P = 0.03) and by 43% after perindopril (P = 1.4 × 10-6) associated with increased DNA methylation (P = 0.04). Immunolabelling confirmed reduced cortical renin after earlier RAS blockade (P = 0.002). RNA sequencing identified differential expression of mRNAs, miRNAs, and lncRNAs with evidence of networking and co-regulation. These included 13 candidate genes (Grhl1, Ammecr1l, Hs6st1, Nfil3, Fam221a, Lmo4, Adamts1, Cish, Hif3a, Bcl6, Rad54l2, Adap1, Dok4), the miRNA miR-145-3p, and the lncRNA AC115371. Gene ontogeny analyses revealed that these networks were enriched with genes relevant to BP, RAS, and the kidneys., Conclusion: Early RAS inhibition in SHR resets genetic pathways and networks resulting in a legacy of reduced Ren expression and BP persisting for a minimum of 6 weeks., Competing Interests: Conflict of interest: There are no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca2+ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca2+ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca2+ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy., (© 2021 Han et al.)

Aims: Angiotensin-converting enzyme 2 (ACE2) is the cellular entry point for severe acute respiratory syndrome coronavirus (SARS-CoV-2)-the cause of coronavirus disease 2019 (COVID-19). However, the effect of renin-angiotensin system (RAS)-inhibition on ACE2 expression in human tissues of key relevance to blood pressure regulation and COVID-19 infection has not previously been reported., Methods and Results: We examined how hypertension, its major metabolic co-phenotypes, and antihypertensive medications relate to ACE2 renal expression using information from up to 436 patients whose kidney transcriptomes were characterized by RNA-sequencing. We further validated some of the key observations in other human tissues and/or a controlled experimental model. Our data reveal increasing expression of ACE2 with age in both human lungs and the kidney. We show no association between renal expression of ACE2 and either hypertension or common types of RAS inhibiting drugs. We demonstrate that renal abundance of ACE2 is positively associated with a biochemical index of kidney function and show a strong enrichment for genes responsible for kidney health and disease in ACE2 co-expression analysis., Conclusion: Our results indicate that neither hypertension nor antihypertensive treatment is likely to alter the expression of the key entry receptor for SARS-CoV-2 in the human kidney. Our data further suggest that in the absence of SARS-CoV-2 infection, kidney ACE2 is most likely nephro-protective but the age-related increase in its expression within lungs and kidneys may be relevant to the risk of SARS-CoV-2 infection., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Hypertrophic cardiomyopathy thickens heart muscles, reducing functionality and increasing risk of cardiac disease and morbidity. Genetic factors are involved, but their contribution is poorly understood. We used the hypertrophic heart rat (HHR), a unique normotensive polygenic model of cardiac hypertrophy and heart failure, to investigate the role of genes associated with monogenic human cardiomyopathy. We selected 42 genes involved in monogenic human cardiomyopathies to study: 1) DNA variants, by sequencing the whole genome of 13-wk-old HHR and age-matched normal heart rat (NHR), its genetic control strain; 2) mRNA expression, by targeted RNA-sequencing in left ventricles of HHR and NHR at 5 ages (2 days old and 4, 13, 33, and 50 wk old) compared with human idiopathic dilated cardiomyopathy data; and 3) microRNA expression, with rat microRNA microarrays in left ventricles of 2-day-old HHR and age-matched NHR. We also investigated experimentally validated microRNA-mRNA interactions. Whole-genome sequencing revealed unique variants mostly located in noncoding regions of HHR and NHR. We found 29 genes differentially expressed in at least 1 age. Genes encoding desmoglein 2 ( Dsg2) and transthyretin ( Ttr) were significantly differentially expressed at all ages in the HHR, but only Ttr was also differentially expressed in human idiopathic cardiomyopathy. Lastly, only two microRNAs differentially expressed in the HHR were present in our comparison of validated microRNA-mRNA interactions. These two microRNAs interact with five of the genes studied. Our study shows that genes involved in monogenic forms of human cardiomyopathies may also influence polygenic forms of the disease.

Background: Among the growing numbers of patients with heart failure, up to one half have heart failure with preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF is a substantial and escalating unmet clinical need-and the lack of HFpEF-specific animal models represents a major preclinical barrier in advancing understanding of HFpEF. As established treatments for heart failure with reduced ejection fraction (HFrEF) have proven ineffective for HFpEF, the contention that the intrinsic cardiomyocyte phenotype is distinct in these 2 conditions requires consideration. Our goal was to validate and characterize a new rodent model of HFpEF, undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology., Methods and Results: The selectively inbred Hypertrophic Heart Rat (HHR) strain exhibits adult cardiac enlargement (without hypertension) and premature death (40% mortality at 50 weeks) compared to its control strain, the normal heart rat. Hypertrophy was characterized in vivo by maintained systolic parameters (ejection fraction at 85%-90% control) with marked diastolic dysfunction (increased E/E'). Surprisingly, HHR cardiomyocytes were hypercontractile, exhibiting high Ca 2+ operational levels and markedly increased L-type Ca 2+ channel current. In HHR, prominent regions of reparative fibrosis in the left ventricle free wall adjacent to the interventricular septum were observed., Conclusions: Thus, the cardiomyocyte remodeling process in the etiology of this HFpEF model contrasts dramatically with the suppressed Ca 2+ cycling state that typifies heart failure with reduced ejection fraction. These findings may explain clinical observations, that treatments considered appropriate for heart failure with reduced ejection fraction are of little benefit for HFpEF-and suggest a basis for new therapeutic strategies., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Background: Cardiac hypertrophy increases the risk of developing heart failure and cardiovascular death. The neutrophil inflammatory protein, lipocalin-2 (LCN2/NGAL), is elevated in certain forms of cardiac hypertrophy and acute heart failure. However, a specific role for LCN2 in predisposition and etiology of hypertrophy and the relevant genetic determinants are unclear. Here, we defined the role of LCN2 in concentric cardiac hypertrophy in terms of pathophysiology, inflammatory expression networks, and genomic determinants., Methods and Results: We used 3 experimental models: a polygenic model of cardiac hypertrophy and heart failure, a model of intrauterine growth restriction and Lcn2 -knockout mouse; cultured cardiomyocytes; and 2 human cohorts: 114 type 2 diabetes mellitus patients and 2064 healthy subjects of the YFS (Young Finns Study). In hypertrophic heart rats, cardiac and circulating Lcn2 was significantly overexpressed before, during, and after development of cardiac hypertrophy and heart failure. Lcn2 expression was increased in hypertrophic hearts in a model of intrauterine growth restriction, whereas Lcn2 -knockout mice had smaller hearts. In cultured cardiomyocytes, Lcn2 activated molecular hypertrophic pathways and increased cell size, but reduced proliferation and cell numbers. Increased LCN2 was associated with cardiac hypertrophy and diastolic dysfunction in diabetes mellitus. In the YFS, LCN2 expression was associated with body mass index and cardiac mass and with levels of inflammatory markers. The single-nucleotide polymorphism, rs13297295, located near LCN2 defined a significant cis -eQTL for LCN2 expression., Conclusions: Direct effects of LCN2 on cardiomyocyte size and number and the consistent associations in experimental and human analyses reveal a central role for LCN2 in the ontogeny of cardiac hypertrophy and heart failure., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Left ventricular (LV) hypertrophy (LVH) is a heritable trait that is common in type 2 diabetes and is associated with the development of heart failure. The transcriptional factor Kruppel like factor 15 (KLF15) is expressed in the heart and acts as a repressor of cardiac hypertrophy in experimental models. This study investigated if KLF15 gene variants were associated with LVH in type 2 diabetes. In stage 1 of a 2-stage approach, patients with type 2 diabetes and no known cardiac disease were prospectively recruited for a transthoracic echocardiographic assessment (Melbourne Diabetes Heart Cohort) (n=318) and genotyping of two KLF15 single nucleotide polymorphisms (SNPs) (rs9838915, rs6796325). In stage 2, the association of KLF15 SNPs with LVH was investigated in the Genetics of Diabetes Audit and Research in Tayside Scotland (Go-DARTS) type 2 diabetes cohort (n=5631). The KLF15 SNP rs9838915 A allele was associated in a dominant manner with LV mass before (P=0.003) and after (P=0.001) adjustment for age, gender, body mass index (BMI) and hypertension, and with adjusted septal (P<0.0001) and posterior (P=0.004) wall thickness. LVH was present in 35% of patients. Over a median follow up of 5.6years, there were 22 (7%) first heart failure hospitalizations. The adjusted risk of heart failure hospitalization was 5.5-fold greater in those with LVH and the rs9838915 A allele compared to those without LVH and the GG genotype (hazard ratio (HR) 5.5 (1.6-18.6), P=0.006). The association of rs9838915 A allele with LVH was replicated in the Go-DARTS cohort. We have identified the KLF15 SNP rs9838915 A allele as a marker of LVH in patients with type 2 diabetes, and replicated these findings in a large independent cohort. Studies are needed to characterize the functional importance of these results, and to determine if the SNP rs9838915 A allele is associated with LVH in other high risk patient cohorts., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Cardiac hypertrophy is the most potent cardiovascular risk factor after age, and relative mortality risk linked with cardiac hypertrophy is greater in women. Ischemic heart disease is the most common form of cardiovascular pathology for both men and women, yet significant differences in incidence and outcomes exist between the sexes. Cardiac hypertrophy and ischemia are frequently occurring dual pathologies. Whether the cellular (cardiomyocyte) mechanisms underlying myocardial damage differ in women and men remains to be determined. In this study, utilizing an in vitro experimental approach, our goal was to examine the proposition that responses of male/female cardiomyocytes to ischemic (and adrenergic) stress may be differentially modulated by the presence of pre-existing cardiac hypertrophy., Methods: We used a novel normotensive custom-derived hypertrophic heart rat (HHR; vs control strain normal heart rat (NHR)). Cardiomyocyte morphologic and electromechanical functional studies were performed using microfluorimetric techniques involving simulated ischemia/reperfusion protocols., Results: HHR females exhibited pronounced cardiac/cardiomyocyte enlargement, equivalent to males. Under basal conditions, a lower twitch amplitude in female myocytes was prominent in normal but not in hypertrophic myocytes. The cardiomyocyte Ca(2+) responses to β-adrenergic challenge differed in hypertrophic male and female cardiomyocytes, with the accentuated response in males abrogated in females-even while contractile responses were similar. In simulated ischemia, a marked and selective elevation of end-ischemia Ca(2+) in normal female myocytes was completely suppressed in hypertrophic female myocytes-even though all groups demonstrated similar shifts in myocyte contractile performance. After 30 min of simulated reperfusion, the Ca(2+) desensitization characterizing the male response was distinctively absent in female cardiomyocytes., Conclusions: Our data demonstrate that cardiac hypertrophy produces dramatically different basal and stress-induced pathophenotypes in female- and male-origin cardiomyocytes. The lower Ca(2+) operational status characteristic of female (vs male) cardiomyocytes comprising normal hearts is not exhibited by myocytes of hypertrophic hearts. After ischemia/reperfusion, availability of activator Ca(2+) is suppressed in female hypertrophic myocytes, whereas sensitivity to Ca(2+) is blunted in male hypertrophic myocytes. These findings demonstrate that selective intervention strategies should be pursued to optimize post-ischemic electromechanical support for male and female hypertrophic hearts.

Background: Low socioeconomic position (SEP) is associated with increased cardiovascular (CV) disease risk, but the relative importance of SEP in childhood and adulthood, and of changes in SEP between these two life stages, remains unclear. Studies of families may help clarify these issues. We aimed to assess whether SEP in young adulthood, or change in SEP from childhood to young adulthood, was associated with five continuously measured CV risk factors., Methods: We used data from 286 adult Australian families from the Victorian Family Heart Study (VFHS), in which some offspring have left home (n = 364) and some remained at home (n = 199). SEP (defined as the Index of Relative Socioeconomic Disadvantage) was matched to addresses. We fitted variance components models to test whether young adult SEP and/or change in SEP was associated with systolic blood pressure, diastolic blood pressure, body mass index (BMI), total cholesterol or high-density lipoprotein cholesterol, after adjustment for parental SEP and within-family correlation., Results: An increase in SEP of 100 SEIFA units from childhood to adulthood was associated with a lower BMI (β = -0.49 kg/m(2), P < 0.01) only., Conclusions: These results suggest that a change in SEP in young adulthood is an important predictor of BMI, independent of childhood SEP., (© The Author 2015. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Short telomeres are associated with increased risk of cardiovascular disease. Here we studied cardiomyocyte telomere length at key ages during the ontogeny of cardiac hypertrophy and failure in the hypertrophic heart rat (HHR) and compared these with the normal heart rat (NHR) control strain. Key ages corresponded with the pathophysiological sequence beginning with fewer cardiomyocytes (2 days), leading to left ventricular hypertrophy (LVH) (13 wk) and subsequently progression to heart failure (38 wk). We measured telomere length, tissue activity of telomerase, mRNA levels of telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc), and expression of the telomeric regulator microRNA miR-34a. Cardiac telomere length was longer in the HHR compared with the control strain at 2 days and 38 wk, but shorter at 13 wk. Neonatal HHR had higher cardiac telomerase activity and expression of Tert and miR-34a. Telomerase activity was not different at 13 or 38 wk. Tert mRNA and Terc RNA were overexpressed at 38 wk, while miR-34a was overexpressed at 13 wk but downregulated at 38 wk. Circulating leukocytes were strongly correlated with cardiac telomere length in the HHR only. The longer neonatal telomeres in HHR are likely to reflect fewer fetal and early postnatal cardiomyocyte cell divisions and explain the reduced total cardiomyocyte complement that predisposes to later hypertrophy and failure. Although shorter telomeres were a feature of cardiac hypertrophy at 13 wk, they were not present at the progression to heart failure at 38 wk., (Copyright © 2016 the American Physiological Society.)

Background: The role of copy number variation (CNV) has been poorly explored in essential hypertension in part due to technical difficulties in accurately assessing absolute numbers of DNA copies. Droplet digital PCR (ddPCR) provides a powerful new approach to CNV quantitation. The aim of our study was to investigate whether CNVs located in regions previously associated with blood pressure (BP) variation in genome-wide association studies (GWAS) were associated with essential hypertension by the use of ddPCR., Methods: Using a "power of extreme" approach, we quantified nucleic acids using ddPCR in white subjects from the Victorian Family Heart Study with extremely high (n = 96) and low (n = 92) SBP, providing power equivalent to 1714 subjects selected at random., Results: A deletion of the CNVs esv27061 and esv2757747 on chromosome 1p13.2 was significantly more prevalent in extreme high BP subjects after adjustment for age, body mass index and sex (12.6% vs. 2.2%; P = 0.013)., Conclusions: Our data suggests that CNVs within regions identified in previous GWAS may play a role in human essential hypertension.

Variants in the gene encoding the γ-subunit of the epithelial sodium channel (SCNN1G) are associated with both Mendelian and quantitative effects on blood pressure. Here, in 4 cohorts of 1611 white European families composed of a total of 8199 individuals, we undertook staged testing of candidate single-nucleotide polymorphisms for SCNN1G (supplemented with imputation based on data from the 1000 Genomes Project) followed by a meta-analysis in all of the families of the strongest candidate. We also examined relationships between the genotypes and relevant intermediate renal phenotypes, as well as expression of SCNN1G in human kidneys. We found that an intronic single-nucleotide polymorphism of SCNN1G (rs13331086) was significantly associated with age-, sex-, and body mass index-adjusted blood pressure in each of the 4 populations (P<0.05). In an inverse variance-weighted meta-analysis of this single-nucleotide polymorphism in all 4 of the populations, each additional minor allele copy was associated with a 1-mm Hg increase in systolic blood pressure and 0.52-mm Hg increase in diastolic blood pressure (SE=0.33, P=0.002 for systolic blood pressure; SE=0.21, P=0.011 for diastolic blood pressure). The same allele was also associated with higher 12-hour overnight urinary potassium excretion (P=0.04), consistent with increased epithelial sodium channel activity. Renal samples from hypertensive subjects showed a nonsignificant (P=0.07) 1.7-fold higher expression of SCNN1G compared with normotensive controls. These data provide genetic and phenotypic evidence in support of a role for a common genetic variant of SCNN1G in blood pressure determination.

The P2X4 receptor is involved in endothelium-dependent changes in large arterial tone in response to shear stress and is, therefore, potentially relevant to arterial compliance and pulse pressure. Four identified nonsynonymous polymorphisms in P2RX4 were reproduced in recombinantly expressed human P2X4. Electrophysiological studies showed that one of these, the Tyr315>Cys mutation (rs28360472), significantly reduced the peak amplitude of the ATP-induced inward current to 10.9% of wild-type P2X4 receptors in transfected HEK-293 cells (10 μmol/L of ATP; n=4-8 cells; P<0.001). Concentration-response curves for ATP and the partial agonist BzATP demonstrate that the 315Cys-P2X4 mutant had an increased EC(50) value for both ligands. Mutation of Tyr315>Cys likely disrupts the agonist binding site of P2X4 receptors, a finding supported by molecular modeling based on the zebrafish P2X4 receptor crystal structure. We tested inheritance of rs28360472 encoding the Tyr315>Cys mutation in P2RX4 against pulse pressure in 2874 subjects from the Victorian Family Heart Study. The minor allele frequency was 0.014 (1.4%). In a variance components analysis we found significant association with pulse pressure (P=0.023 for total association) where 1 minor allele increased pulse pressure by 2.84 mm Hg (95% CI: 0.41-5.27). This increase in pulse pressure associated with inheritance of 315Cys-P2X4 receptors might reflect reduced large arterial compliance as a result of impaired endothelium-dependent vasodilation in large arteries.

Objectives: The aim of this study was to examine the relationship between erectile problems in men and cardiovascular disease (CVD) mortality., Background: Although there are plausible mechanisms linking erectile dysfunction (ED) with coronary heart disease (CHD) and stroke, studies are scarce., Methods: In a cohort analysis of the ADVANCE (Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified-Release Controlled Evaluation) trial population, 6,304 men age 55 to 88 years with type 2 diabetes participated in a baseline medical examination when inquiries were made about ED. Over 5 years of follow-up, during which study members attended repeat clinical examinations, the presence of fatal and nonfatal CVD outcomes, cognitive decline, and dementia was ascertained., Results: After adjusting for a range of covariates, including existing illness, psychological health, and classic CVD risk factors, relative to those who were free of the condition, baseline ED was associated with an elevated risk of all CVD events (hazard ratio: 1.19; 95% confidence interval: 1.08 to 1.32), CHD (hazard ratio: 1.35; 95% confidence interval: 1.16 to 1.56), and cerebrovascular disease (hazard ratio: 1.36; 95% confidence interval: 1.11 to 1.67). Men who experienced ED at baseline and at 2-year follow-up had the highest risk for these outcomes., Conclusions: In this cohort of men with type 2 diabetes, ED was associated with a range of CVD events., (Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Diabetes imposes a substantial burden globally in terms of premature mortality, morbidity, and health care costs. Estimates of economic outcomes associated with diabetes are essential inputs to policy analyses aimed at prevention and treatment of diabetes. Our objective was to estimate and compare event rates, hospital utilization, and costs associated with major diabetes-related complications in high-, middle-, and low-income countries., Methods and Findings: Incidence and history of diabetes-related complications, hospital admissions, and length of stay were recorded in 11,140 patients with type 2 diabetes participating in the Action in Diabetes and Vascular Disease (ADVANCE) study (mean age at entry 66 y). The probability of hospital utilization and number of days in hospital for major events associated with coronary disease, cerebrovascular disease, congestive heart failure, peripheral vascular disease, and nephropathy were estimated for three regions (Asia, Eastern Europe, and Established Market Economies) using multiple regression analysis. The resulting estimates of days spent in hospital were multiplied by regional estimates of the costs per hospital bed-day from the World Health Organization to compute annual acute and long-term costs associated with the different types of complications. To assist, comparability, costs are reported in international dollars (Int$), which represent a hypothetical currency that allows for the same quantities of goods or services to be purchased regardless of country, standardized on purchasing power in the United States. A cost calculator accompanying this paper enables the estimation of costs for individual countries and translation of these costs into local currency units. The probability of attending a hospital following an event was highest for heart failure (93%-96% across regions) and lowest for nephropathy (15%-26%). The average numbers of days in hospital given at least one admission were greatest for stroke (17-32 d across region) and heart failure (16-31 d) and lowest for nephropathy (12-23 d). Considering regional differences, probabilities of hospitalization were lowest in Asia and highest in Established Market Economies; on the other hand, lengths of stay were highest in Asia and lowest in Established Market Economies. Overall estimated annual hospital costs for patients with none of the specified events or event histories ranged from Int$76 in Asia to Int$296 in Established Market Economies. All complications included in this analysis led to significant increases in hospital costs; coronary events, cerebrovascular events, and heart failure were the most costly, at more than Int$1,800, Int$3,000, and Int$4,000 in Asia, Eastern Europe, and Established Market Economies, respectively., Conclusions: Major complications of diabetes significantly increase hospital use and costs across various settings and are likely to impose a high economic burden on health care systems.

Buffering of blood pressure during change of posture such as standing is controlled largely by the baroreflex. In our population-based Victorian Family Heart Study (VFHS), we previously demonstrated that, on average, systolic blood pressure (SBP) changes very little on standing; however, interindividual variation is substantial and shows familial aggregation, with approximately 25% of the variance attributable to genetic factors. Our genomewide linkage analysis suggests a region on chromosome 12p that harbors two strong candidate genes, KCNJ8 and ABCC9, encoding the channel-forming inward rectifier subunit Kir6.1 and the ATP-sensitive binding cassette SUR2B, respectively. These are key components of smooth muscle ATP-sensitive potassium (K(ATP)) channels, important regulators of arterial tone and blood flow and central to autonomic baroreceptor control of changes in total peripheral resistance. We performed a comprehensive association analysis of 47 tag single nucleotide polymorphisms (SNPs) spanning the KCNJ8 and ABCC9 gene regions with postural change in SBP (DeltaSBP). To augment power, we took a selective genotyping approach in which we compared allele and genotype frequencies between 150 unrelated individuals with high (positive) DeltaSBP (> or = 7 mmHg) and 150 unrelated individuals with low (negative) DeltaSBP (< or = -7 mmHg) drawn from the offspring generation (18-30 yr) of the VFHS. Association analyses showed that no SNPs demonstrated statistically significant differences in genotype frequencies between groups, particularly after adjustments for multiple testing. We conclude that sequence variants in KCNJ8 and ABCC9 are unlikely to contribute to variation in DeltaSBP. Other genes in the identified chromosome 12p region warrant investigation.

The relative importance of various blood pressure indices on cardiovascular risk in people with type 2 diabetes mellitus has not been established. This study compares the strengths of the associations between different baseline blood pressure variables (systolic blood pressure [SBP], diastolic blood pressure [DBP], pulse pressure [PP], and mean arterial pressure) and the 4.3-year risk of major cardiovascular events in the Action in Diabetes and Vascular Disease: Preterax and Diamicron-Modified Release Controlled Evaluation Study. Mean (SD) age for the 11 140 participants was 65.8 years (6.4 years). During follow-up, 1000 major cardiovascular events, 559 major coronary events, and 468 cardiovascular deaths were recorded. After adjustment for age, sex, and treatment allocation, the hazard ratios (95% CIs) associated with 1 increment in SD for the risk of major cardiovascular events were 1.17 (1.10 to 1.24) for SBP; 1.20 (1.13 to 1.28) for PP; 1.12 (1.05 to 1.19) for mean arterial pressure; and 1.04 (0.98 to 1.11) for DBP. The areas under the receiver operating characteristic curve were slightly higher for SBP and PP compared with mean arterial pressure and DBP for major cardiovascular and coronary events. Using achieved instead of baseline blood pressure values marginally improved the effect estimates for SBP, DBP, and mean arterial pressure, with no significant differences in the areas under the receiver operating characteristic curve between models with SBP and those with PP. In conclusion, SBP and PP are the 2 best and DBP is the least effective determinant of the risk of major cardiovascular outcomes in the relatively old patients with type 2 diabetes mellitus participating in the Action in Diabetes and Vascular Disease: Preterax and Diamicron-Modified Release Controlled Evaluation Study. However, SBP may be the simplest and most useful predictor across a wider range of age groups and populations.

Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases, but the proximal triggers for autophagy are unknown. Angiotensin II plays a central role in the pathogenesis of cardiac hypertrophy and heart failure. In this study, we used angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptor-expressing adenoviruses in cultured neonatal cardiomyocytes to provide the first demonstration that neonatal cardiomyocyte autophagic activity is differentially modulated by AT(1) and AT(2) receptor subtypes. Angiotensin II stimulation (48 hours) of neonatal cardiomyocytes expressing the AT(1) receptor alone (Ad-AT(1); 10 multiplicities of infection) induced a significant increase in the number of HcRed-LC3 autophagosomes per cell (17.3+/-1.6 versus 33.3+/-4.1 autophagosomes per cell; P<0.05). Coexpression of a high ratio of AT(2):AT(1) (Ad-AT(2):Ad-AT(1) multiplicity of infection ratio: 20:5) receptors completely abrogated the AT(1)-mediated increase in autophagy (9.3+/-1.4 versus 33.3+/-4.1 autophagosomes per cell; P<0.05). Treatment with the AT(2) receptor antagonist PD123319 did not reverse the AT(2)-mediated antiautophagic effect. AT(1)- and AT(2)-mediated autophagic responses were also assessed in cardiomyocytes from a genetic model that exhibits neonatal myocardial growth suppression. In these neonate myocyte cultures, AT(1) receptor activation induced a marked increase in the number of myocytes containing cytoplasmic vacuoles compared with the control (22.7+/-4.1% versus 1.1+/-0.6%; P<0.001) and was characterized by a nonapoptotic autophagic phenotype. The incidence of cardiomyocyte autophagic vacuolization in this myocyte population decreased dramatically to only 0.4+/-0.2% in myocytes infected with a high ratio of Ad-AT(2):Ad-AT(1). This study provides the first description of reciprocal regulation of cardiomyocyte autophagic induction by the AT(1) and AT(2) receptor subtypes.

The identification of genetic factors influencing cardiac growth independently of increased load is crucial to an understanding of the molecular and cellular basis of pathological cardiac hypertrophy. The central aim of this investigation was to determine how pathological hypertrophy in the adult can be linked with disturbances in cardiomyocyte growth and viability in early neonatal development. The hypertrophic heart rat (HHR) model is derived from the spontaneously hypertensive rat and exhibits marked cardiac hypertrophy, in the absence of a pressure load at maturity. Hearts were harvested from male HHR, and control strain normal heart rats (NHR), at different stages of postnatal development [neonatal (P2), 4 wk, 6 wk, 8 wk, 12 wk, 20 wk]. Isolated neonatal cardiomyocytes were prepared to evaluate cell size, number, and binucleation. At postnatal day 2, HHR hearts were considerably smaller than control NHR (4.3 +/- 0.2 vs. 5.0 +/- 0.1 mg/g, P < 0.05). Cardiac growth restriction in the neonatal HHR was associated with reduced myocyte size (length and width) and an increased proportion of binucleated cardiomyocytes. Furthermore, the number of cardiomyocytes isolated from HHR neonatal hearts was significantly less ( approximately 29%) than NHR. We also observe that growth stress in the neonate is associated with accentuated PI3K and suppressed MAPK activation, although these signaling pathways are normalized in the adult heart exhibiting established hypertrophy. Thus, using the HHR model, we identified novel molecular and cellular mechanisms involving premature exit from the cell cycle, reduced cardiomyocyte endowment, and dysregulated trophic signaling during early development, which are implicated in the etiology of heritable cardiac hypertrophy in the adult.

Background: The functional polymorphism that explains the established association of the androgen receptor (AR) with androgenetic alopecia (AGA) remains unidentified, but Copy Number Variation (CNV) might be relevant. CNV involves changes in copy number of large segments of DNA, leading to the altered dosage of gene regulators or genes themselves. Two recent reports indicate regions of CNV in and around AR, and these have not been studied in relation to AGA. The aim of this preliminary case-control study was to determine if AR CNV is associated with AGA, with the hypothesis that CNV is the functional AR variant contributing to this condition., Methodology/principal Findings: Multiplex Ligation-dependent Probe Amplification was used to screen for CNV in five AR exons and a conserved, non-coding region upstream of AR in 85 men carefully selected as cases and controls for maximal phenotypic contrast. There was no evidence of CNV in AR in any of the cases or controls, and thus no evidence of significant association between AGA and AR CNV., Conclusions/significance: The results suggest this form of genomic variation at the AR locus is unlikely to predispose to AGA.

Important sex differences in cardiovascular disease outcomes exist, including conditions of hypertrophic cardiomyopathy and cardiac ischemia. Studies of sex differences in the extent to which load-independent (primary) hypertrophy modulates the response to ischemia-reperfusion (I/R) damage have not been characterized. We have previously described a model of primary genetic cardiac hypertrophy, the hypertrophic heart rat (HHR). In this study the sex differences in HHR cardiac function and responses to I/R [compared to control normal heart rat (NHR)] were investigated ex vivo. The ventricular weight index was markedly increased in HHR female (7.82 +/- 0.49 vs. 4.80 +/- 0.10 mg/g; P < 0.05) and male (5.76 +/- 0.22 vs. 4.62 +/- 0.07 mg/g; P < 0.05) hearts. Female hearts of both strains exhibited a reduced basal contractility compared with strain-matched males [maximum first derivative of pressure (dP/dt(max)): NHR, 4,036 +/- 171 vs. 4,258 +/- 152 mmHg/s; and HHR, 3,974 +/- 160 vs. 4,540 +/- 259 mmHg/s; P < 0.05]. HHR hearts were more susceptible to I/R (I = 25 min, and R = 30 min) injury than NHR hearts (decreased functional recovery, and increased lactate dehydrogenase efflux). Female NHR hearts exhibited a significantly greater recovery (dP/dt(max)) post-I/R relative to male NHR (95.0 +/- 12.2% vs. 60.5 +/- 9.4%), a resistance to postischemic dysfunction not evident in female HHR (29.0 +/- 5.6% vs. 25.9 +/- 6.3%). Ventricular fibrillation was suppressed, and expression levels of Akt and ERK1/2 were selectively elevated in female NHR hearts. Thus the occurrence of load-independent primary cardiac hypertrophy undermines the intrinsic resistance of female hearts to I/R insult, with the observed abrogation of endogenous cardioprotective signaling pathways consistent with a potential mechanistic role in this loss of protection.

Systolic blood pressure is determined in large part by genes. Six independent studies have reported evidence of linkage between systolic pressure and chromosome 16p12 that incorporates SCNN1G, the gene encoding the gamma-subunit of the epithelial sodium channel. We undertook the first comprehensive association analysis of SCNN1G and systolic pressure. To achieve genetic contrast, we sampled unrelated subjects within the upper (mean: 166 mm Hg; n=96) and lower (mean: 98 mm Hg; n=94) 10% of the systolic pressure distribution of 2911 subjects from the Victorian Family Heart Study. We examined genotypes and haplotypes related to 26 single nucleotide polymorphisms across SCNN1G and its promoter. Each of 3 single nucleotide polymorphisms (rs13331086, rs11074553, and rs4299163) in introns 5 and 6 showed evidence of association with systolic pressure in logistic regression analyses adjusted for age, sex, and body mass index. Considered as a haplotype block, these single nucleotide polymorphisms were significantly associated with systolic pressure (haplo.score global: P=0.0001). In permutation analyses to account for multiple testing, a result such as this was observed only once in 10,000 permutations. The estimated frequency of 1 haplotype (TGC) was substantially greater in high (13.3%) than low (0.6%) systolic pressure subjects (P=0.0001). Three other haplotypes (TGG, TAC, and GGC) showed associations with high or low systolic pressure consistent with the observed associations of their composite alleles. These findings identify relatively common polymorphisms in the SCNN1G gene that are associated with high systolic blood pressure in the general Australian white population.

Androgenetic alopecia, or male pattern baldness, is a complex condition with a strong heritable component. In 2001, we published the first significant evidence of a genetic association between baldness and a synonymous coding SNP (rs6152) in the androgen receptor gene, AR. Recently, this finding was replicated in three independent studies, confirming an important role for AR in the baldness phenotype. In one such replication study, it was claimed that the causative variant underlying the association was likely to be the polyglycine (GGN) repeat polymorphism, one of two apparently functional triplet repeat polymorphisms located in the exon 1 transactivating domain of the gene. Here, we extend our original association finding and present comprehensive evidence from approximately 1,200 fathers and sons drawn from 703 families of the Victorian Family Heart Study, a general population Caucasian cohort, that neither exon 1 triplet repeat polymorphism is causative in this condition. Seventy-eight percent of fathers (531/683) and 30% of sons (157/520) were affected to some degree with AGA. We utilised statistical methods appropriate for the categorical nature of the phenotype and familial structure of the cohort, and determined that whilst SNP rs6152 was strongly associated with baldness (P < 0.0001), the GGN triplet repeat was not (P = 0.13). In the absence of any other known common functional coding variants, we argue that the causative variant is likely to be in the non-coding region, and yet to be identified. The identification of functional non-coding variants surrounding AR may have significance not only for baldness, but also for the many other complex conditions that have thus far been linked to AR.

There have been a number of genome-wide linkage studies for adult height in recent years. These studies have yielded few well-replicated loci, and none have been further confirmed by the identification of associated gene variants. The inconsistent results may be attributable to the fact that few studies have combined accurate phenotype measures with informative statistical modelling in healthy populations. We have performed a multi-stage genome-wide linkage analysis for height in 275 adult sibling pairs drawn randomly from the Victorian Family Heart Study (VFHS), a healthy population-based Caucasian cohort. Height was carefully measured in a standardised fashion on regularly calibrated equipment. Following genome-wide identification of a peak Z-score of 3.14 on chromosome 3 at 69 cM, we performed a fine-mapping analysis of this region in an extended sample of 392 two-generation families. We used a number of variance components models that incorporated assortative mating and shared environment effects, and we observed a peak LOD score of approximately 3.5 at 78 cM in four of the five models tested. We also demonstrated that the most prevalent model in the literature gave the worst fit, and the lowest LOD score (2.9) demonstrating the importance of appropriate modelling. The region identified in this study replicates the results of other genome-wide scans of height and bone-related phenotypes, strongly suggesting the presence of a gene important in bone growth on chromosome 3p. Association analyses of relevant candidate genes should identify the genetic variants responsible for the chromosome 3p linkage signal in our population.

Lower plasma levels of high-density lipoprotein cholesterol (HDL-C) are associated with the metabolic syndrome (insulin resistance, obesity, hypertension) and higher cardiovascular risk. Recent association studies have suggested rare alleles responsible for very low HDL-C levels. However, for individual cardiovascular risk factors, the majority of population-attributable deaths are associated with average rather than extreme levels. Therefore, genetic factors that determine the population variation of HDL-C are particularly relevant. We undertook genome-wide and fine mapping to identify linkage to HDL-C in healthy adult nuclear families from the Victorian Family Heart Study. In 274 adult sibling pairs (average age 24 years, average plasma HDL-C 1.4 mmol/l), genome-wide mapping revealed suggestive evidence for linkage on chromosome 4 (Z score = 3.5, 170 cM) and nominal evidence for linkage on chromosomes 1 (Z = 2.1, 176 cM) and 6 (Z = 2.6, 29 cM). Using genotypes and phenotypes from 932 subjects (233 of the sibling pairs and their parents), finer mapping of the locus on chromosome 4 strengthened our findings with a peak probability (Z score = 3.9) at 169 cM. Our linkage data suggest that chromosome 4q32.3 is linked with normal population variation in HDL-C. This region coincides with previous reports of linkage to apolipoprotein AII (a major component of HDL) and encompasses the gene encoding the carboxypeptidase E, relevant to the metabolic syndrome and HDL-C. These findings are relevant for further understanding of the genetic determinants of cardiovascular risk at a population level.