Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

Background: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD’s actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). Aim/method: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. Results: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. Conclusion: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.

Myocardial infarction in the C57BL/6J mouse - A quantifiable and highly reproducible experimental model

Introduction: The laboratory mouse is a powerful tool in cardiovascular research. In this report, we describe a method for a reproducible mouse myocardial infarction model that would allow subsequent comparative and quantitative studies on molecular and pathophysiological variables. Methods: (A) The distribution of the major coronary arteries including the septal artery in the left ventricle of the C57BL/6J mice (n=20) was mapped by perfusion of latex dye or fluorescent beads through the aorta. (B) The territory of myocardial infarction after the ligation of the most proximal aspect of the left anterior descending (LAD) coronary artery was quantified. (C) The consistency in the histological changes parallel to the infarction at different time points was analyzed. Results: (A) The coronary artery tree of the mouse is different from human and, particularly, in regard to the blood supply of the septum. (B) Contrary to previous belief, the septal coronary artery in the mouse is variable in origin. (C) A constant ligation of the LAD immediately below the left auricular level ensures a statistically significant reproducible infarct size. (D) The ischemic changes can be monitored at a histological level in a way similar to what is described in the human. Conclusion: We illustrate a method for maximal reproducibility of experimental acute myocardial infarction in the mouse model, due to a consistent loss of perfusion in the lower half of the left ventricle. This will allow the study of molecular and physiological variables in a controlled and quantifiable experimental model environment. (C) 2004 Elsevier Inc. All rights reserved.

The effect of multiple micronutrient supplementation on left ventricular ejection fraction in patients with chronic stable heart failure: a randomized, placebo-controlled trial:A randomized, placebo-controlled trial

Objectives: This study sought to investigate the effect of a multiple micronutrient supplement on left ventricular ejection fraction (LVEF) in patients with heart failure. Background: Observational studies suggest that patients with heart failure have reduced intake and lower concentrations of a number of micronutrients. However, there have been very few intervention studies investigating the effect of micronutrient supplementation in patients with heart failure. Methods: This was a randomized, double-blind, placebo-controlled, parallel-group study involving 74 patients with chronic stable heart failure that compared multiple micronutrient supplementation taken once daily versus placebo for 12 months. The primary endpoint was LVEF assessed by cardiovascular magnetic resonance imaging or 3-dimensional echocardiography. Secondary endpoints were Minnesota Living With Heart Failure Questionnaire score, 6-min walk test distance, blood concentrations of N-terminal prohormone of brain natriuretic peptide, C-reactive protein, tumor necrosis factor alpha, interleukin-6, interleukin-10, and urinary levels of 8-iso-prostaglandin F2 alpha. Results: Blood concentrations of a number of micronutrients increased significantly in the micronutrient supplement group, indicating excellent compliance with the intervention. There was no significant difference in mean LVEF at 12 months between treatment groups after adjusting for baseline (mean difference: 1.6%, 95% confidence interval: -2.6 to 5.8, p = 0.441). There was also no significant difference in any of the secondary endpoints at 12 months between treatment groups. Conclusions: This study provides no evidence to support the routine treatment of patients with chronic stable heart failure with a multiple micronutrient supplement. (Micronutrient Supplementation in Patients With Heart Failure [MINT-HF]; NCT01005303).

Progression of left atrial volume index in a population at risk for heart failure:a substudy of the STOP-HF (St Vincent's Screening TO Prevent Heart Failure) trial

AIMS: Limited data are available concerning the evolution of the left atrial volume index (LAVI) in pre-heart failure (HF) patients. The aim of this study was to investigate clinical characteristics and serological biomarkers in a cohort with risk factors for HF and evidence of serial atrial dilatation.

METHODS AND RESULTS: This was a prospective substudy within the framework of the STOP-HF cohort (NCT00921960) involving 518 patients with risk factors for HF electively undergoing serial clinical, echocardiographic, and natriuretic peptide assessment. Mean follow-up time between assessments was 15 ± 6 months. 'Progressors' (n = 39) were defined as those with serial LAVI change ≥3.5 mL/m(2) (and baseline LAVI between 20 and 34 mL/m(2)). This cut-off was derived from a calculated reference change value above the biological, analytical, and observer variability of serial LAVI measurement. Multivariate analysis identified significant baseline clinical associates of LAVI progression as increased age, beta-blocker usage, and left ventricular mass index (all P < 0.05). Serological biomarkers were measured in a randomly selected subcohort of 30 'Progressors' matched to 30 'Non-progressors'. For 'Progressors', relative changes in matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinase 1 (TIMP1), and the TIMP1/MMP9 ratio, markers of interstitial remodelling, tracked with changes in LAVI over time (all P < 0.05).

CONCLUSION: Accelerated LAVI increase was found to occur in up to 14% of all pre-HF patients undergoing serial echocardiograms over a relatively short follow-up period. In a randomly selected subcohort of 'Progressors', changes in LAVI were closely linked with alterations in MMP9, TIMP1, and the ratio of these enzymes, a potential aid in highlighting this at-risk group.

Influence of progressive renal dysfunction in chronic heart failure

Chronic heart failure (CHF) is often associated with impaired renal function due to hypoperfusion. Such patients are very sensitive to changes in renal perfusion pressure, and may develop acute tubular necrosis if the pressure falls too far. The situation is complicated by the use of diuretics, ACE inhibitors and spironolactone, all of which may affect renal function and potassium balance. Chronic renal failure (CRF) may also be associated with fluid overload. Anaemia and hypertension in CRF contribute to the development of left ventricular hypertrophy (LVH), which carries a poor prognosis, so correction of these factors is important.

Unintended cardiac irradiation during left-sided breast cancer radiotherapy

Objective: Cardiac irradiation during left-sided breast radiotherapy may lead to
deleterious cardiac side effects. Using image guided radiotherapy, it is possible
to exclude the heart from treatment fields and monitor reproducibility of virtual simulation (VS) fields at treatment delivery using electronic portal imaging (EPI). Retrospectively, we evaluate the incidence of cardiac irradiation at VS and subsequent unintended cardiac irradiation during treatment.

Methods: Patients receiving left-sided radiotherapy to the breast or chest wall,
treated with a glancing photon field technique during a four-month period, were
included. VS images and EPIs during radiotherapy delivery were visually assessed.
The presence of any portion of the heart within the treatment field at VS or during treatment was recorded. Central lung distance and maximum heart distance were recorded.

Results: Of 128 patients, 45 (35.1%) had any portion of the heart within the
planned treatment field. Of these, inclusion of the heart was clinically unavoidable in 25 (55.6%). Of those with no heart included in the treatment fields at VS, 41 (49.4%) had presence of the heart as assessed on EPI during treatment.

Conclusion: Unintended cardiac irradiation during left-sided breast radiotherapy treatment occurs in a sizeable proportion of patients.

Advances in knowledge: Despite the use of three-dimensional computed tomography simulation and cardiac shielding, sizeable proportions of patients receiving left-sided breast cancer radiotherapy have unintended cardiac irradiation.

Volumetric modulated arc therapy and breath-hold in image-guided locoregional left-sided breast irradiation

PURPOSE: To investigate the effects of using volumetric modulated arc therapy (VMAT) and/or voluntary moderate deep inspiration breath-hold (vmDIBH) in the radiation therapy (RT) of left-sided breast cancer including the regional lymph nodes.

MATERIALS AND METHODS: For 13 patients, four treatment combinations were compared; 3D-conformal RT (i.e., forward IMRT) in free-breathing 3D-CRT(FB), 3D-CRT(vmDIBH), 2 partial arcs VMAT(FB), and VMAT(vmDIBH). Prescribed dose was 42.56 Gy in 16 fractions. For 10 additional patients, 3D-CRT and VMAT in vmDIBH only were also compared.

RESULTS: Dose conformity, PTV coverage, ipsilateral and total lung doses were significantly better for VMAT plans compared to 3D-CRT. Mean heart dose (D(mean,heart)) reduction in 3D-CRT(vmDIBH) was between 0.9 and 8.6 Gy, depending on initial D(mean,heart) (in 3D-CRT(FB) plans). VMAT(vmDIBH) reduced the D(mean,heart) further when D(mean,heart) was still >3.2 Gy in 3D-CRT(vmDIBH). Mean contralateral breast dose was higher for VMAT plans (2.7 Gy) compared to 3DCRT plans (0.7 Gy).

CONCLUSIONS: VMAT and 3D-CRT(vmDIBH) significantly reduced heart dose for patients treated with locoregional RT of left-sided breast cancer. When Dmean,heart exceeded 3.2 Gy in 3D-CRT(vmDIBH) plans, VMAT(vmDIBH) resulted in a cumulative heart dose reduction. VMAT also provided better target coverage and reduced ipsilateral lung dose, at the expense of a small increase in the dose to the contralateral breast.

The heart of a dragon: 3D anatomical reconstruction of the 'scaly-foot gastropod' (Mollusca: Gastropoda: Neomphalina) reveals its extraordinary circulatory system

Introduction: The 'scaly-foot gastropod' (Chrysomallon squamiferum Chen et al., 2015) from deep-sea hydrothermal vent ecosystems of the Indian Ocean is an active mobile gastropod occurring in locally high densities, and it is distinctive for the dermal scales covering the exterior surface of its foot. These iron-sulfide coated sclerites, and its nutritional dependence on endosymbiotic bacteria, are both noted as adaptations to the extreme environment in the flow of hydrogen sulfide. We present evidence for other adaptations of the 'scaly-foot gastropod' to life in an extreme environment, investigated through dissection and 3D tomographic reconstruction of the internal anatomy.

Results: Our anatomical investigations of juvenile and adult specimens reveal a large unganglionated nervous system, a simple and reduced digestive system, and that the animal is a simultaneous hermaphrodite. We show that Chrysomallon squamiferum relies on endosymbiotic bacteria throughout post-larval life. Of particular interest is the circulatory system: Chrysomallon has a very large ctenidium supported by extensive blood sinuses filled with haemocoel. The ctenidium provides oxygen for the host but the circulatory system is enlarged beyond the scope of other similar vent gastropods. At the posterior of the ctenidium is a remarkably large and well-developed heart. Based on the volume of the auricle and ventricle, the heart complex represents approximately 4 % of the body volume. This proportionally giant heart primarily sucks blood through the ctenidium and supplies the highly vascularised oesophageal gland. Thus we infer the elaborate cardiovascular system most likely evolved to oxygenate the endosymbionts in an oxygen poor environment and/or to supply hydrogen sulfide to the endosymbionts.

Conclusions: This study exemplifies how understanding the autecology of an organism can be enhanced by detailed investigation of internal anatomy. This gastropod is a large and active species that is abundant in its hydrothermal vent field ecosystem. Yet all of its remarkable features-protective dermal sclerites, circulatory system, high fecundity-can be viewed as adaptations beneficial to its endosymbiont microbes. We interpret these results to show that, as a result of specialisation to resolve energetic needs in an extreme chemosynthetic environment, this dramatic dragon-like species has become a carrying vessel for its bacteria.

Characteristics, complications, and gaps in evidence-based interventions in rheumatic heart disease: the Global Rheumatic Heart Disease Registry (the REMEDY study)

AIMS: Rheumatic heart disease (RHD) accounts for over a million premature deaths annually; however, there is little contemporary information on presentation, complications, and treatment.

METHODS AND RESULTS: This prospective registry enrolled 3343 patients (median age 28 years, 66.2% female) presenting with RHD at 25 hospitals in 12 African countries, India, and Yemen between January 2010 and November 2012. The majority (63.9%) had moderate-to-severe multivalvular disease complicated by congestive heart failure (33.4%), pulmonary hypertension (28.8%), atrial fibrillation (AF) (21.8%), stroke (7.1%), infective endocarditis (4%), and major bleeding (2.7%). One-quarter of adults and 5.3% of children had decreased left ventricular (LV) systolic function; 23% of adults and 14.1% of children had dilated LVs. Fifty-five percent (n = 1761) of patients were on secondary antibiotic prophylaxis. Oral anti-coagulants were prescribed in 69.5% (n = 946) of patients with mechanical valves (n = 501), AF (n = 397), and high-risk mitral stenosis in sinus rhythm (n = 48). However, only 28.3% (n = 269) had a therapeutic international normalized ratio. Among 1825 women of childbearing age (12-51 years), only 3.6% (n = 65) were on contraception. The utilization of valvuloplasty and valve surgery was higher in upper-middle compared with lower-income countries.

CONCLUSION: Rheumatic heart disease patients were young, predominantly female, and had high prevalence of major cardiovascular complications. There is suboptimal utilization of secondary antibiotic prophylaxis, oral anti-coagulation, and contraception, and variations in the use of percutaneous and surgical interventions by country income level.

Serum Amyloid P-Component Prevents Cardiac Remodeling in Hypertensive Heart Disease

The potential for serum amyloid P-component (SAP) to prevent cardiac remodeling and identify worsening diastolic dysfunction (DD) was investigated. The anti-fibrotic potential of SAP was tested in an animal model of hypertensive heart disease (spontaneously hypertensive rats treated with SAP [SHR - SAP] × 12 weeks). Biomarker analysis included a prospective study of 60 patients with asymptomatic progressive DD. Compared with vehicle-treated Wistar-Kyoto rats (WKY-V), the vehicle-treated SHRs (SHR-V) exhibited significant increases in left ventricular mass, perivascular collagen, cardiomyocyte size, and macrophage infiltration. SAP administration was associated with significantly lower left ventricular mass (p < 0.01), perivascular collagen (p < 0.01), and cardiomyocyte size (p < 0.01). Macrophage infiltration was significantly attenuated in the SHR-SAP group. Biomarker analysis showed significant decreases in SAP concentration over time in patients with progressive DD (p < 0.05). Our results indicate that SAP prevents cardiac remodeling by inhibiting recruitment of pro-fibrotic macrophages and that depleted SAP levels identify patients with advancing DD suggesting a role for SAP therapy.