Scott Brouilette

Modulated inflammation by injection of high-mobility group box 1 recovers post-infarction chronically failing heart.

Background— Inflammation plays an important role in the progress of adverse ventricular remodeling after myocardial infarction. High-mobility group box 1 (HMGB1) is a nuclear protein, which has recently been uncovered to also act as a modifier of inflammation when released. We hypothesized that HMGB1 injection could preferentially modulate local myocardial inflammation, attenuate ventricular remodeling, and subsequently improve cardiac performance of postinfarction chronic heart failure. Methods and Results— Three weeks after left coronary artery ligation, HMGB1 (2.5 &mgr;g) or PBS was intramyocardially injected into rat hearts. At 28 days after injection, left ventricular ejection fraction was significantly improved after HMGB1 injection compared to PBS (39.3±1.4 versus 33.3±1.8%; P<0.01). Accumulation of CD45+ inflammatory cells, two thirds of which were OX62+ dendritic cells, in the peri-infarct area was significantly attenuated by HMGB1 injection. Dramatic changes in the expression of major proinflammatory cytokines were not detected by microarray or RT-PCR. Adverse ventricular remodeling including cardiomyocyte hypertrophy (cardiomyocyte cross-sectional area; 439±7 versus 458±6 &mgr;m2; P<0.05) and extracellular collagen deposition (collagen volume fraction; 11.9±0.4 versus 15.2±0.6%; P<0.01) was attenuated by HMGB1 injection. Analyses of signal transduction pathways revealed that HMGB1 injection activated ERK1/2, but not p38, Akt, and Smad3. Cardiac regeneration and neovascularization were not observed. Conclusion— HMGB1 injection modulated the local inflammation in the postinfarction chronically failing myocardium, particularly via reducing the accumulation of dendritic cells. This modulated inflammation resulted in attenuated fibrosis and cardiomyocyte hypertrophy, which thereby improved global cardiac function. These data suggest that HMGB1 may be valuable for the chronic heart failure treatment.

Donor cell-type specific paracrine effects of cell transplantation for post-infarction heart failure

Cell transplantation is an emerging therapy for treating post-infarction heart failure. Although the paracrine effect has been proposed to be an important mechanism for the therapeutic benefits, details remain largely unknown. This study compared various aspects of the paracrine effect after transplantation of either bone marrow mononuclear cells (BMC) or skeletal myoblasts (SMB) into the post-infarction chronically failing heart. Three weeks after left coronary artery ligation, adult rats received intramyocardial injection of either BMC, SMB or PBS only. Echocardiography demonstrated that injection of either cell type improved cardiac function compared to PBS injection. Interestingly, BMC injection markedly improved neovascularization in the border areas surrounding infarcts, while SMB injection decreased fibrosis in both the border and remote areas. Injection of either cell type similarly reduced hypertrophy of cardiomyocytes as assessed by cell-size planimetry using isolated cardiomyocytes. Quantitative RT-PCR revealed that, among 15 candidate mediators of paracrine effects studied, Fgf2 and Hgf were upregulated only after BMC injection, while Mmp2 and Timp4 were modulated after SMB injection. Additional investigations of signalling pathways relevant to heart failure by western blotting showed that p38 and STAT3 were temporarily activated after BMC injection, in contrast, ERK1/2 and JNK were activated after SMB injection. There was no difference in activation of Akt, PKD or Smad3 among groups. These data suggest that paracrine effects observed after cell transplantation in post-infarction heart failure were noticeably different between cell types in terms of mediators, signal transductions and consequent effects.

A simple and novel method for RNA-seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase.

Background: Deep sequencing of single cell‐derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA‐seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5‐mediated library preparation, which brings high quality libraries, is likely one of the solutions. Results and Conclusions: Here, we tested the applicability of hyperactive Tn5‐mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA‐seq results were comparable to the conventional method. Thus, this Tn5‐mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Developmental Dynamics 241:1584–1590, 2012.

Telomere shortening and telomerase activity in ischaemic cardiomyopathy patients – Potential markers of ventricular arrhythmia

BACKGROUND Implantable cardioverter defibrillators (ICDs) reduce mortality in patients with ischaemic cardiomyopathy at high risk of ventricular arrhythmias (VA). However, the current indication for ICD prescription needs improvement. Telomere and telomerase in leucocytes have been shown to associate with biological ageing and pathogenesis of cardiovascular diseases. We hypothesised that leucocyte telomere length, load-of-short telomeres and/or telomerase activity are associated with VA occurrence in ischaemic cardiomyopathy patients. METHODS AND RESULTS 90 ischaemic cardiomyopathy patients with primary prevention ICDs were recruited. 35 had received appropriate therapy from the ICD for potentially-fatal VA while the remaining 55 patients had not. No significant differences in baseline demographic data relevant to telomere biology were seen between the two groups. There was no significant difference in the age and sex adjusted mean telomere length analysed by qPCR between the groups (p=0.88). In contrast, the load-of-short telomeres assessed by Universal-STELA method and telomerase activity by TRAP assay were both higher in patients who had appropriate ICD therapy and were significantly associated with incidence of ICD therapy (p=0.02, p=0.02). ROC analyses demonstrated that the sensitivity and specificity of these telomere dynamics in predicting potentially-fatal VA was higher than the current gold-standard - left ventricular ejection fraction (AUC 0.82 versus 0.47). CONCLUSION The load-of-short telomeres and telomerase activity had a significant association with ICD therapy (for VA) in ischaemic cardiomyopathy patients. These biomarkers should be tested in prospective studies to assess their clinical utility in predicting VA after myocardial infarction and guiding primary prevention ICD prescription.

Current Genomics in Cardiovascular Medicine

Cardiovascular disease (CVD) is a heterogeneous, complex trait that has a major impact on human morbidity and mortality. Common genetic variation may predispose to common forms of CVD in the community, and rare genetic conditions provide unique pathogenetic insights into these diseases. With the advent of the Human Genome Project and the genomic era, new tools and methodologies have revolutionised the field of genetic research in cardiovascular medicine. In this review, we describe the rationale for the current emphasis on large-scale genomic studies, elaborate on genome wide association studies and summarise the impact of genomics on clinical cardiovascular medicine and how this may eventually lead to new therapeutics and personalised medicine.

Association of genetic variation in telomere-related SNP and telomerase with ventricular arrhythmias in ischemic cardiomyopathy.

Telomeres are known to provide genomic stability and telomere length has been associated with cardiovascular diseases. Moreover, a higher telomerase activity has been shown to be associated with ventricular arrhythmias (VA) in ischemic cardiomyopathy. Increasing evidence suggests that genetic variation in key telomere genes has an impact on telomerase activity. Each copy of the minor allele of SNP rs12696304, at a locus including TERC (telomerase), has been associated with ∼75 base pairs reduction in mean telomere length likely mediated by an effect on TERC expression. We investigated the impact of genetic variation of this SNP on telomerase and its association with VA in ischemic cardiomyopathy patients.

69 Association of TERC Related Genetic Variation and Telomerase Activity with Ventricular Arrhythmias in Ischaemic Cardiomtyopathy

Introduction Implantable cardioverter defibrillators (ICD) reduce mortality in ischaemic cardiomyopathy patients at a high risk of ventricular arrhythmias (VA), which are the commonest cause of sudden death. However, ICDs are associated with morbidity and mortality. Importantly 67% of patients never receive an appropriate shock after ICD implantation under current guidelines, suggesting a need for better risk stratification. Telomere and telomerase activity (TA) in leukocytes have recently been shown to correlate with biological ageing and pathogenesis of cardiovascular diseases. Telomerase maintains telomere length and is composed of reverse transcriptase TERT and RNA template TERC. Evidence suggests that genetic variation in key genes has a key impact on TA. Association of SNP12696304 on chromosome 3q26 (at a locus that includes TERC) with telomere length has been shown. We investigated the association between genetic variation in SNP12696304 and leukocyte TA with incidence of VA in ischaemic cardiomyopathy patients. Methods 90 ischaemic cardiomyopathy Caucasian patients with primary prevention ICDs were recruited. Genomic DNA was isolated from venous blood samples. TA was measured by the telomere repeat amplification protocol and genotyping done using Taqman SNP assay. Continuous data were compared by unpaired T-test and categorical data by Chi-square test. Logistic regression was used to determine correlation of genotype and TA with VA. Results There was no significant difference in baseline demographics between patients with VA(Cases) and no-VA(Controls) as shown in Table 1. Abstract 69 Table 1 Baseline demographics N=90 Cases Controls p-value Number 35 55 Age, mean±SD 69.3 ± 7.6 71.6 ± 8.8 0.19 WCC, mean±SD 7.7 ± 1.7 7.46 ± 1.7 0.43 Male, n (%) 32 (91.4) 48 (87.3) 0.78 LVEF,%, mean±SD 26.73 ± 7.2 27.24 ± 7.6 0.75 eGFR, mean±SD 63.94 ± 21.8 60.59 ± 20.22 0.49 Follow up since ICD implant, mthsmean±SD 46.5 ± 26.9 38.1 ± 30.5 0.18 The TA was significantly higher in the cases and correlated with incidence of VA (p-value 0.02). No significant correlation between the genotype and VA was identified (C/C OR 1; C/G OR 0.54, CI 0.21 – 1.417, p-value 0.343; G/G OR 0.80, CI 0.198 – 3.236, p-value 0.907). There was a significant correlation between risk of VA and TA increase in C/C genotype (OR7.5, CI1–56.6, p-value 0.04). This correlation was not significant in the G/G or C/G genotype as shown in Figure 1. Abstract 69 Figure 1 Telomerase activity across genotypes Conclusion There is a significant correlation between TA and VA in ischaemic cardiomyopathy patients. Moreover, homozygosity for C allele of SNP 12696304 (encoding TERC) significantly effects telomerase expression. Thus, increased TA predisposes individuals with C/C genotype to a higher incidence of VA. TA in this genotype can be developed as a biomarker to predict VA and guide ICD prescription in ischaemic cardiomyopathy.

Differentiation-dependent telomeric long non-coding transcription in a model of skeletal myogenesis

Telomeres comprise the distal ends of eukaryotic chromosomes, serve to maintain genomic integrity and are extended by the ribonucleoprotein telomerase. Recent evidence indicates that telomeres are transcribed to generate long non-coding RNAs (lncRNAs) and that these transcripts (TERRA) may inhibit telomerase activity. In this study we assessed telomerase activity and telomeric lncRNA expression in a mouse model of skeletal myogenesis. Using the C2C12 cell line we demonstrated decreased telomerase activity during differentiation into terminally-differentiated skeletal myotubes. Despite existing in a post-mitotic state, residual telomerase activity remained in C2C12 myotubes, indicating a role independent of telomere extension. Telomeric transcripts were detected in both myoblasts and myotubes, with reduced expression during differentiation correlating with reduced telomerase expression. Our data indicate that in a mouse model of skeletal myogenesis TERRA expression does not reduce telomerase activity, suggesting that their relationship is more complex than originally perceived; the role of telomeric derived lncRNAs in relation to telomerase activity may be cell-type specific. These findings raise the possibility for novel non-telomerase regulatory function for TERRA-lncRNAs during skeletal myogenesis.

056 Leucocyte telomere/telomerase dynamics in patients with implantable cardioverter defibrillator; potential biomarker for ventricular arrhythmias

Introduction Implantable cardioverter defibrillators (ICDs) have been shown to reduce mortality in patients with ischaemic cardiomyopathy at a high risk of ventricular arrhythmias (VA), which are the commonest cause of sudden death. However, ICDs are associated with morbidity and mortality Importantly 67% of patients never receive an appropriate shock after ICD implantation under the current indication, suggesting a need for better risk stratification tools. Telomere and telomerase in leucocytes have recently been shown to correlate with biological aging, health status, and also with pathogenesis/prognosis of various cardiovascular diseases. We hypothesise that the leucocyte telomere length and/or telomerase activity correlate with the incidence of VA in ischaemic cardiomyopathy patients. Methods 73 ischaemic cardiomyopathy Caucasian patients with primary prevention ICDs were recruited to this retrospective study between October 2010 and January 2011 at St Bartholomews Hospital. Concentrated leucocyte fraction was obtained from venous blood sample of recruited patients and stored at −80°C in an anonymous manner. Genomic DNA was extracted from these and telomere length measured by telomere PCR. Each telomere length was expressed as a ratio (telomere length: single copy gene). Telomerase activity was measured using Roche Telo TAAGGG ELISA kit. All samples were analysed in duplicate and investigators were semi-blinded to arrhythmia history. Continuous data were compared using unpaired t test and categorical data by χ2 test. Logistic regression analysis was performed to determine if telomere length/telomerase activity independently predict the likelihood of a shock (fatal VA). A probability value of p<0.05 was defined as significant. Results There were no significant differences between the Shock (patients received appropriate shocks from ICD; n=25) and Non-shock (patients received no shock; n=48) groups in terms of baseline demographics as shown in Abstract 056 table 1. There was no significant difference in the age, sex and WCC adjusted telomere length between the Shock and Non-shock groups (p=0.439). Expected age and WCC turnover related telomere attrition (p=0.031 and 0.031) was observed. In contrast, telomerase activity was significantly higher in the Shock group thank in the Non-shock group (0.5682 vs 0.2105) and co-related to the incidence of shock (p=0.01). This did not appear to be related to an acute response associated with VA.Abstract 056 Table 1 Group1 (shock) Group 2 (non-shock) p Value Number of patients 25 48 Age, mean (range) 70.8 (56–87) 70.8 (46–87) 0.97 Female gender (%) 4 16.67 0.15 Diabetes (%) 5 (20) 14 (29.2) 0.57 Hypertension (%) 5 (20) 14 (29.2) 0.57 Time since implant (months), mean 50.68 34.13 0.06 LVEF 27.64 27.29 0.84 Conclusion This is the first study to characterise the telomere dynamics of patients at high risk of sudden cardiac death and co-relate this with the incidence of VA. Leucocyte telomerase activity independently predicted the likelihood of shock in ischaemic cardiomyopathy patients with primary prevention ICDs. Thus leucocyte telomerase activity may be a potential biomarker for prediction of fatal arrhythmia and guide ICD prescription. To validate these results, a prospective study is now ongoing.